WO2020057645A1 - 新型白介素2及其用途 - Google Patents

新型白介素2及其用途 Download PDF

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WO2020057645A1
WO2020057645A1 PCT/CN2019/107054 CN2019107054W WO2020057645A1 WO 2020057645 A1 WO2020057645 A1 WO 2020057645A1 CN 2019107054 W CN2019107054 W CN 2019107054W WO 2020057645 A1 WO2020057645 A1 WO 2020057645A1
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mutein
cells
amino acid
sequence
seq
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PCT/CN2019/107054
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English (en)
French (fr)
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康立山
付凤根
周帅祥
史新震
刘军建
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信达生物制药(苏州)有限公司
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Priority to CA3098765A priority Critical patent/CA3098765A1/en
Priority to EP19861566.8A priority patent/EP3854805A4/en
Priority to US17/059,539 priority patent/US20210213102A1/en
Priority to CN201980029372.8A priority patent/CN112105634B/zh
Priority to JP2020571636A priority patent/JP2022501009A/ja
Priority to AU2019344875A priority patent/AU2019344875B2/en
Publication of WO2020057645A1 publication Critical patent/WO2020057645A1/zh
Priority to JP2023142152A priority patent/JP2023174651A/ja

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Definitions

  • the invention relates to a novel interleukin 2 (IL-2) mutant protein and use thereof.
  • the present invention relates to compounds having improved properties compared to wild-type IL-2, such as improved drugability, reduced IL-2R ⁇ receptor binding ability, and / or increased IL-2R ⁇ receptor binding ability.
  • IL-2 mutein The invention also provides a fusion protein comprising the IL-2 mutein, an immunoconjugate, and a nucleic acid encoding the IL-2 mutein, a vector comprising the nucleic acid, and a host cell.
  • the invention further provides a method for preparing the IL-2 mutein, a pharmaceutical composition comprising the IL-2 mutein, and a therapeutic use of the mutein.
  • Interleukin-2 also known as T cell growth factor (TCGF)
  • TCGF T cell growth factor
  • TCGF T cell growth factor
  • human IL-2 (uniprot: P60568) is synthesized as a precursor peptide of 153 amino acids. After removing the 20 amino acids at the N-terminus, it produces mature secreted IL-2.
  • Interleukin 2 has four antiparallel, amphiphilic alpha helices. These four alpha helices form a quaternary structure that is essential for its function (Smith, Science 240, 1169-76 (1988); Bazan, Science 257, 410-413 (1992)). In most cases, IL-2 works through three different receptors: interleukin 2 receptor alpha (IL-2R ⁇ ; CD25), interleukin 2 receptor beta (IL-2R ⁇ ; CD122), and interleukin 2 receptor gamma ( IL-2R ⁇ ; CD132).
  • IL-2R ⁇ interleukin 2 receptor alpha
  • IL-2R ⁇ interleukin 2 receptor beta
  • IL-2R ⁇ interleukin 2 receptor gamma
  • IL-2R ⁇ and IL-2R ⁇ are essential for IL-2 signaling, while IL-2R ⁇ (CD25) is not necessary for signaling, but can confer high affinity binding of IL-2 to receptors (Krieg et al., Proc Natl Acad Sci 107, 11906-11 (2010)).
  • the trimer receptor (IL-2 ⁇ ) formed by the combination of IL-2R ⁇ , ⁇ , and ⁇ is the IL-2 high affinity receptor (KD about 10 pM), and the dimer receptor (IL- 2 ⁇ ) is an intermediate affinity receptor (KD about 1 nM), and an IL-2 receptor formed by an ⁇ subunit alone is a low affinity receptor.
  • Immune cells express dimer or trimer IL-2 receptors. Dimer receptors are expressed on cytotoxic CD8 + T cells and natural killer cells (NK), while trimer receptors are mainly on activated lymphocytes and CD4 + CD25 + FoxP3 + inhibitory regulatory T cells (Treg) Expression (Byman, O. and Sprent. J. Nat. Rev. Immunol. 12, 180-190 (2012)). Because resting effector T cells and NK cells do not have CD25 on the cell surface, they are relatively insensitive to IL-2. Treg cells consistently express the highest level of CD25 in the body. Therefore, under normal circumstances, IL-2 will preferentially stimulate Treg cell proliferation.
  • IL-2 mediates multiple roles in the immune response by binding to the IL-2 receptor on different cells.
  • IL-2 can stimulate T cell proliferation and differentiation, induce the production of cytotoxic T lymphocytes (CTL), promote B cell proliferation and differentiation and immunoglobulin synthesis, and stimulate natural killer (NK)
  • CTL cytotoxic T lymphocytes
  • NK natural killer
  • IL-2 can promote the maintenance of immunosuppressive CD4 + CD25 + regulatory T cells (ie, Treg cells) (Fontenot et al., Nature Immunol 6,1142-51 (2005); D'Cruz and Klein, Nature Immunol 6,1152-59 (2005); Maloy and Powrie, Nature Immunol 6,1171-72 (2005)), and mediate activation-induced cell death (AICD) and participate in the establishment of immune tolerance against autoantigens and tumor antigens And maintenance (Lenardo et al., Nature 353: 858 (1991)), thereby causing tumor resistance caused by AICD and immunosuppression by activated Treg cells in patients.
  • Ie immunosuppressive CD4 + CD25 + regulatory T cells
  • AICD mediate activation-induced cell death
  • IL-2 induces pulmonary edema by directly binding to the IL-2 trimer receptor (IL-2 ⁇ ) on lung endothelial cells (Krieg et al., Proc Nat Acad Sci USA 107, 11906-11 (2010)).
  • Rodrigo Vazquez-Lombardi et al. (Nature Communications, 8: 15373, DOI: 10.1038 / ncomms15373) proposed a triple mutant human IL-2 mutein IL-2 3X , which has amino acid residue positions 38, 43 and 61, respectively. Residue mutations R38D-K43E-E61R caused the mutant protein to not bind to IL-2R ⁇ , but the mutant protein had a weak effect on activating CD25 - cells, and the bias of activation on CD25 + cells still remained. In addition, Rodrigo Vazquez-Lombardi et al. Also proposed to improve the pharmacodynamic properties of interleukins by preparing interleukin 2-Fc fusions, but the expression of the fusion protein is low and it is easy to form aggregates.
  • the present invention satisfies the above-mentioned needs by providing a new IL-2 mutein with improved drug-forming properties and / or improved IL-2 receptor selectivity / biasing relative to wild-type IL-2.
  • the invention provides novel IL-2 muteins.
  • the IL-2 muteins of the invention have one or more of the following characteristics:
  • the invention provides an IL-2 mutein comprising an introduced mutant glycosylation motif at the binding interface of IL-2 and IL-2R ⁇ ; in other embodiments, the invention provides an IL-2
  • the B'C 'loop region comprises an IL-2 mutein that is deleted and / or replaced with a shortened loop sequence; in yet other embodiments, the invention provides a glycosylation motif with a mutation and a shortened B'C 'Loop sequence of both IL-2 muteins.
  • the present invention provides a fusion protein and an immunoconjugate, a pharmaceutical composition, and a combination product comprising the IL-2 mutein; a nucleic acid encoding the IL-2 mutein, a vector and a host cell comprising the nucleic acid; Invented methods of IL-mutant proteins, fusion proteins, and immunoconjugates.
  • the present invention also provides a method for treating diseases using the IL-2 mutein and fusions and immunoconjugates of the present invention, and a method and use for stimulating the immune system of a subject.
  • the methods of the invention result in strong activation and expansion of CD25 - effector T cells and NK cells in a subject.
  • the method of the present invention can effectively reduce the immune down-regulation effect of IL-2 on Treg cells.
  • Figure 1 shows the crystal structures of IL-2 and IL-2R ⁇ (PDB: 1Z92) (A) and the schematic diagram of the IL-2 glycosylation-modified protein (B).
  • Figure 2 shows the IL-2 crystal structure (PBD: 2ERJ) (A) and the B'C'loop structure of human and mouse IL-2 and human IL15 superpose (B).
  • Fig. 3 shows the HPLC purity detection pattern of the purified sample of IL-2R ⁇ .
  • Figure 4 shows the HPLC purity test pattern of the purified sample of IL-2R ⁇ .
  • Figure 5 shows the CD8 + CD25 construct selected and some of the IL-2 mutant -FC - / CD25 + T-cell activation signal curves of p-STAT5.
  • Figure 6 shows the mature protein sequence (SEQ ID NO: 26) of human interleukin (IL-2) and its amino acid residue numbering, and shows exemplary IL-2 glycosylation mutants and IL-2 chimeric and A truncated B'C 'loop mutant.
  • IL-2 human interleukin
  • the term “comprising” or “including” means including the recited elements, integers, or steps, but does not exclude any other elements, integers, or steps.
  • the terms “comprising” or “including” are used, unless otherwise indicated, the case of consisting of the mentioned elements, integers, or steps is also covered.
  • an IL-2 mutein that "comprises” or “includes” a certain mutation or combination of mutations, it is also intended to encompass an IL-2 mutein having only that mutation or combination of mutations.
  • the wild-type "interleukin-2” or "IL-2” refers to a parent IL-2 protein as a template for introducing a mutation or a combination of mutations of the present invention, preferably a naturally occurring IL-2 protein, for example, derived from human, Natural IL-2 proteins of mice, rats, and non-human primates, including unprocessed (e.g., signal peptide removed) and processed (e.g., signal peptide removed) forms.
  • a full-length natural human IL-2 sequence containing a signal peptide is shown in SEQ ID NO: 29, and the sequence of its mature protein is shown in SEQ ID NO: 30.
  • the expression also includes naturally occurring IL-2 allelic and splice variants, isotypes, homologs, and species homologs.
  • the expression also includes variants of natural IL-2, for example, the variants can have at least 95% -99% or higher identity with natural IL-2 or have no more than 1-10 or 1-5 amino acids Mutations (especially conservative amino acid substitutions) and have substantially the same IL-2R ⁇ binding affinity and / or IL2R ⁇ binding affinity as the native IL-2 protein.
  • wild-type IL-2 may contain amino acid mutations that do not affect its binding to the IL-2 receptor compared to the native IL-2 protein, for example, natural human IL with mutation C125S introduced at position 125 -2 protein (uniprot: P60568) belongs to the wild-type IL-2 of the present invention.
  • natural human IL with mutation C125S introduced at position 125 -2 protein belongs to the wild-type IL-2 of the present invention.
  • An example of a wild-type human IL-2 protein containing a C125S mutation is shown in SEQ ID NO: 26.
  • the wild-type IL-2 sequence may have at least 85%, 95%, or even at least 96%, 97%, 98%, or 99% or more of the amino acid sequence of SEQ ID NO: 26 or 29 or 30. High amino acid sequence identity.
  • amino acid mutations can be amino acid substitutions, deletions, insertions, and additions. Any combination of substitutions, deletions, insertions, and additions can be made to obtain the final mutein construct with the desired properties (eg, reduced IL-2R ⁇ binding affinity).
  • Amino acid deletions and insertions include deletions and insertions at the amino and / or carboxy terminus of a polypeptide sequence. For example, alanine residues can be deleted at full-length human IL-2 position 1.
  • the preferred amino acid mutation is an amino acid substitution. In other embodiments, the preferred amino acid mutation is an amino acid deletion.
  • mutations are introduced at specific mutant amino acid positions described herein to obtain an IL-2 mutein with an altered glycosylation motif. In some embodiments, mutations are introduced at specific mutated amino acid positions described herein to obtain an IL-2 mutein with a shortened B'C 'loop sequence.
  • the amino acid position in the IL-2 protein or polypeptide is the amino acid position numbered according to SEQ ID NO: 26.
  • F42 it refers to the phenylalanine residue F at position 42 of SEQ ID NO: 26, or an amino acid residue corresponding to a corresponding position on another IL-2 polypeptide sequence.
  • Amino acid substitutions are expressed as [original amino acid residues / positions / substituted amino acid residues].
  • amino acid substitution at position 35 to asparagine (N) can be expressed as 35N, and if the original amino acid residue at position 35 is lysine, it can also be expressed as K35N.
  • substituted residue is represented by X, for example, 36X, it means that the amino acid at position 36 can be replaced by any residue. If X has a specific residue value, the position is replaced by the defined specific X residue.
  • the "percent sequence identity" can be determined by comparing the two best aligned sequences within a comparison window.
  • sequence identity is determined over the entire length of a reference sequence (e.g. SEQ ID NO: 26).
  • Sequence alignment methods for comparison are well known in the art. Algorithms suitable for determining percent sequence identity include, for example, the BLAST and BLAST 2.0 algorithms (see Altschul et al., Nuc. Acids Res. 25: 3389-402, 1977 and Altschul et al. J. Mol. Biol. 215: 403-10, 1990. Software available for BLAST analysis available through the public at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). For the purposes of this application, identity The percentage is usually determined using the BLAST 2.0 algorithm set as the default parameter.
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the biological function of the protein / polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • a typical conservative amino acid substitution refers to the replacement of one amino acid with another amino acid having similar chemical properties, such as charge or hydrophobicity.
  • Each of the following six groups contains amino acids that can be conservatively substituted with each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E ); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L) , Methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), and tryptophan (W).
  • the wild-type IL-2 protein may have conservative amino acid substitutions relative to one of SEQ ID NO: 26, 29, or 30, or only conservative amino acid substitutions.
  • the mutant IL-2 protein of the present invention may have conservative amino acid substitutions, or only conservative amino acid substitutions, relative to the IL-2 mutant protein sequence (eg, any of SEQ ID NO: 31-50) specifically given herein. .
  • Binding affinity can be used to reflect the intrinsic binding capacity of interactions between members of a binding pair.
  • the affinity of molecule X for its binding partner Y can be expressed by the equilibrium dissociation constant (K D ), which is the ratio of the dissociation rate constant and the association rate constant (k dis and k on, respectively ).
  • K D equilibrium dissociation constant
  • Binding affinity can be measured by common methods known in the art. A specific method for measuring affinity is the biofilm interference (BLI) technique determination herein.
  • an antibody binding molecule is a polypeptide molecule that can specifically bind an antigen, for example, an immunoglobulin molecule, an antibody or an antibody fragment, such as a Fab fragment and a scFv fragment.
  • an antibody Fc fragment refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region, and may include natural sequence Fc fragments and variant Fc fragments.
  • the human IgG heavy chain Fc fragment extends from the heavy chain Cys226 or from Pro230 to the carboxy terminus.
  • the C-terminal lysine (Lys447) of the Fc-fragment may or may not be present.
  • the Fc fragment may comprise a mutation, such as a L234A / L235A mutation.
  • the numbering of amino acid residues in Fc fragments is based on the EU numbering system, also known as the EU index, such as Kabat, EA, etc., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes Health, Bethesda, MD (1991), NIH Publication 91-3242.
  • the invention provides, in one aspect, a novel IL-2 mutein that has improved pharmaceutical properties and / or improved IL-2 receptor selectivity / preference.
  • the IL-2 protein interacts with the IL-2 receptor to trigger signaling and function.
  • Wild-type IL-2 shows different affinity for different IL-2 receptors.
  • IL-2 ⁇ and ⁇ receptors with lower affinity to wild-type IL-2 are expressed on resting effector cells, including CD8 + cytotoxic T cells and NK cells.
  • IL-2R ⁇ with high affinity to wild-type IL-2 is expressed on regulatory T cells (Treg) cells and activated effector cells. Due to high affinity, wild-type IL-2 preferentially binds to IL-2R ⁇ on the cell surface, then recruits IL-2R ⁇ , releases downstream p-STAT5 signals through IL-2R ⁇ , and stimulates Treg cells and activated effector cells.
  • reducing or eliminating the affinity of IL-2 for the IL-2R ⁇ receptor will reduce the bias of IL-2 preferentially activating CD25 + cells and reduce the immune downregulation of Treg cells mediated by IL-2 effect.
  • maintaining or enhancing affinity for the IL-2 ⁇ receptor will retain or enhance the activation of IL-2 on effector cells such as CD8 + cytotoxic T cells and NK cells, and thus the immune stimulation of IL-2 effect.
  • the inventors have discovered that the expression and / or purity of IL-2 mutein can be improved and / or reduced by introducing one or more specific N glycosylation motifs at the IL-2 and IL-2R ⁇ receptor binding interface. Binding of IL-2 mutein to IL-2R ⁇ .
  • the inventors have also discovered that the IL-2 itself can be replaced by a short B'C 'loop sequence from other interleukin cytokines such as IL-15, or by The B'C 'loop sequence is truncated to increase the expression and / or purity of IL-2 and at the same time increase its affinity for IL-2R ⁇ .
  • the present invention provides an IL-2 mutein having improved properties.
  • the IL-2 mutein of the present invention may have improved properties relative to wild-type IL-2 selected from, for example, one or more of the following: (i) improved expression and / or purity when expressed in mammalian cells (Ii) reduced or eliminated binding to the IL-2R ⁇ receptor; and / or (iii) enhanced binding to the IL-2R ⁇ receptor.
  • the IL-2 mutein of the invention has improved properties relative to wild-type IL-2 selected from, for example, one or more of the following:
  • the IL-2 mutein of the present invention has the properties of the above (1), preferably further has one or more, especially all properties selected from the group consisting of (3) and (5)-(8). ; More preferably still has one or more selected from (2) and (9)-(12), especially all properties.
  • the IL-2 mutein of the present invention has the properties of (2) above, preferably further has one or more, especially all properties selected from (9) to (12); more preferably It further has one or more properties selected from (1), (3) and (5)-(8), especially all properties.
  • the IL-2 muteins of the invention have one or more of the following properties relative to wild-type IL-2: Reduced IL-2 mutein mediated by binding to the high affinity receptor IL-2 ⁇ In vivo toxicity.
  • the IL-2 mutein of the invention has improved pharmaceutical properties, for example, when expressed in mammalian cells, such as H293T cells, preferably when expressed as an Fc fusion protein, has an item selected from the group consisting of A number of properties: (i) better expression than wild-type IL-2 protein; (ii) better stability than wild-type IL-2 protein; and (iii) easy purification to higher protein purity.
  • the IL-2 mutein of the invention exhibits an increased expression level compared to wild IL-2.
  • increased expression occurs in a mammalian cell expression system.
  • the expression level can be determined by any suitable method that allows quantitative or semi-quantitative analysis of the amount of recombinant IL-2 protein in the cell culture supernatant, preferably the supernatant after one-step affinity chromatography purification.
  • the amount of recombinant IL-2 protein in a sample can be assessed by Western blotting or ELISA.
  • the IL-2 mutein of the invention is increased in mammalian cells by at least 1.1-fold, or at least 1.5-fold, or at least 2-fold, 3-fold, or 4 compared to wild-type IL-2. Times more.
  • the IL-2 mutein-Fc fusions of the present invention exhibit more relative to wild-type IL-2 protein fusions Good stability, for example, has less tendency to form aggregates.
  • protein purity is detected by SEC-HPLC technology.
  • the purity of the IL-2 mutein product of the present invention can reach 70%, or 80%, or more than 90%.
  • the IL-2 mutein of the invention reduces the binding affinity of the IL-2R ⁇ receptor by at least 5-fold relative to wild-type IL-2 (eg, IL-2 WT shown in SEQ ID NO: 26), At least 10 times, or at least 25 times, especially at least 30 times, 50 times, or more than 100 times. In a preferred embodiment, the muteins of the invention do not bind the IL-2 receptor alpha.
  • the binding affinity can be determined by biofilm interference (BLI) technology of the IL-2 mutein of the present invention, for example, the IL-2 mutein of the present invention fused to an Fc fragment, and the equilibrium dissociation constant (K of the IL-2R ⁇ receptor). D ) to determine.
  • the monovalent binding affinity of an IL-2 mutein eg, in the form of an Fc fusion
  • a receptor IL-2R ⁇ or IL-2R ⁇ is determined by BLI technology.
  • the binding affinity of the IL-2 mutein of the invention to the IL-2R ⁇ receptor is increased at least 5-fold relative to wild-type IL-2 (eg, IL-2 WT shown in SEQ ID NO: 26), At least 10 times, or at least 25 times, especially at least 30 times, 50 times or 100 times, more preferably at least 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, or 500 times Or more than 550 times.
  • wild-type IL-2 eg, IL-2 WT shown in SEQ ID NO: 26
  • the binding affinity can be determined by the biofilm interference (BLI) technique of the IL-2 mutein of the present invention, for example, the IL-2 mutein of the present invention fused to an Fc fragment, and the equilibrium dissociation constant of the receptor IL-2R ⁇ receptor (K D ) to determine.
  • BLI biofilm interference
  • the IL-2 mutein of the invention binds monovalently to the receptor IL-2R ⁇ receptor.
  • Affinity K D value is less than 10.0E-07M, such as 8.0E-07M to 1.0E-07M, such as 4.0E-07M, 3.0E-07M, 2.0E-07M, 1.0E-07M, and more preferably less than 10.0E-08M For example, less than 9.0E-10M.
  • the IL-2 mutein of the invention results in reduced activation and proliferation of CD25 + cells mediated by IL-2 relative to wild-type IL-2.
  • the CD25 + cells are CD25 + CD8 + T cells.
  • the CD25 + cells are Treg cells.
  • STAT5 phosphorylation assay by detecting IL-2 muteins in CD25 + cells, activation of STAT5 phosphorylation signal to identify IL-2 muteins ability of CD25 + cells activation.
  • the STAT5 phosphorylation in cells can be analyzed by flow cytometry to determine the half-maximum effective concentration (EC50).
  • an IL-2 mutein of the invention results in maintained or enhanced CD25 - effector cell activation and proliferation mediated by IL-2 relative to wild-type IL-2.
  • the ability of an IL-2 mutein to activate CD25 - cells is identified by detecting the EC50 value of an IL-2 mutein that activates a STAT5 phosphorylation signal in CD25 - cells.
  • an IL-2 mutein of the invention activates CD25 + cells relative to a wild-type IL-2 protein (eg, human IL-2 of SEQ ID NO: 26), as determined in a STAT5 phosphorylation assay.
  • the ability is increased by at least 1 time, such as 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times.
  • the IL-2 mutein of the invention removes or reduces the bias of IL-2 to preferential activation of CD25 + cells relative to wild-type IL-2.
  • the CD25 + cells are CD25 + CD8 + T cells.
  • the CD25 + cells are Treg cells.
  • STAT5 phosphorylation assay by detecting IL-2 muteins were in CD25 - and activated EC50 values STAT5 phosphorylation signal cells in the CD25 + cells to identify IL-2 mutein activation CD25 - Cell capacity.
  • IL-2 muteins determining activation of CD25 + cells bias.
  • the mutant protein's bias towards CD25 + is reduced by at least 10-fold, preferably at least 100-fold, 150-fold, or 200-fold relative to the wild-type protein.
  • the invention provides an IL-2 mutein comprising a mutant glycosylation motif at the IL-2 and IL-2Ra binding interface.
  • polypeptides are typically glycosylated via N-linking or O-linking.
  • N-linked glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X-serine (N, X) and asparagine-X-threonine (N, X) are N-linked glycosylation motifs, where X is any other than proline Amino acid. The presence of any of these tripeptide sequences in a polypeptide will result in a potential glycosylation site.
  • an N-linked glycosylation site can be conveniently accomplished by altering the amino acid sequence so that it contains one or more of the aforementioned tripeptide sequences.
  • an N-linked glycosylation site can be added by changing the codon for a single amino acid.
  • a codon encoding N-X-z (where z is any amino acid) may be altered to encode N-X-T (or N-X-S), or a codon encoding y-X-T / S may be altered to encode N-X-T / S.
  • the codons encoding two amino acids can be changed simultaneously to introduce an N-linked glycosylation site (eg, the codons for y-X-z can be changed to encode N-X-T / S).
  • the glycosylation motif that appears in the IL-2 protein due to the introduced mutation can be described as a mutant glycosylation motif.
  • the mutant glycosylation motif K35N-L36-T37 is an N-linked glycosylation motif formed by replacing lysine at position 35 with asparagine and leaving residues at positions 36 and 37 unchanged.
  • the introduced mutant glycosylation motif is an N-linked glycosylation motif, N-X-S / T, where X is any amino acid except proline.
  • X may be the same amino acid as the amino acid corresponding position of wild-type IL-2, or a conservative substitution residue thereof.
  • the present invention provides an IL-2 glycosylated mutein compared to wild-type IL-2 (preferably human IL-2, more preferably IL-2 comprising the SEQ ID NO: 26 sequence), said The mutein contains at least one mutation that introduces one or more glycosylation motifs NXS / T at an amino acid position selected from:
  • the number of N-linked glycosylation sites introduced may be more than one, such as two glycosylation sites.
  • Different glycosylation sites can confer different properties on IL-2, for example, some glycosylation sites can confer improved expression and / or purification properties, and some glycosylation sites can improve IL-2 receptor selectivity .
  • the mutein of the present invention may also contain at least 1-30 amino acid residues different from wild-type IL-2, such as 1-20,1- 15, 1-10, or 1-5 different amino acid residues. These different residues can be conservative substitutions or other mutations that impart other improved properties to IL-2.
  • mutating the glycosylation motif improves the drug-forming properties of the IL-2 protein, and particularly promotes the expression and / or purification of the IL-2 protein.
  • the mutant glycosylation motif that improves the pharmaceutical properties is selected from: 35N-36X-37T / S; 38N-39X-40T / S; and 74N-75X-76T / S.
  • the mutant glycosylation motif is selected from: (i) K35N-L36-T37; (ii) R38N-M39-L40S; and (iii) Q74N-S75-K76T.
  • the mutant glycosylation motif is K35N-L36-T37.
  • the present invention provides, compared to wild-type IL-2, an IL-2 mutein comprising a mutant glycosylation motif selected from: 35N-36X -37T / S; 38N-39X-40T / S; and 74N-75X-76T / S, and the mutant protein has improved pharmaceutical properties.
  • the IL-2 mutein when expressed in mammalian cells, preferably as an Fc fusion protein, can promote the expression and / or purification of the mutein.
  • the mutation may promote the stability of IL-2, for example when expressed as an Fc fusion protein has a reduced tendency to form aggregates during production compared to wild-type IL-2.
  • the mutein can have a higher purity than the wild-type protein.
  • the mutein comprises a mutant glycosylation motif selected from the group consisting of: (i) K35N-L36-T37; and (ii) R38N-M39-L40S compared to wild-type IL-2; (iii) Q74N-S75-K76T; more preferably the mutein comprises a mutant glycosylation motif K35N-L36-T37.
  • the mutant glycosylation motif is introduced into the IL-2 protein by mutating K35N.
  • the present invention provides an IL-2 mutein having a mature region in the amino acid sequence with a wild-type IL-2 protein listed in one of SEQ ID NO: 26, 29, or 30 A mature region that is at least 90% identical and has amino acid residues T37 and a mutation K35N.
  • the mutant glycosylation motif is introduced into the IL-2 protein by a pair of mutations selected from R38N / L40S or Q74N / K76T.
  • the present invention provides an IL-2 mutein having a mature region in the amino acid sequence with a wild-type IL-2 protein listed in one of SEQ ID NO: 26, 29, or 30 Mature region that is at least 90% identical and has a pair of mutations selected from R38N / L40S or Q74N / K76T.
  • the mutein comprises at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of an amino acid sequence selected from the group consisting of SEQ ID NO: 31, 32, and 38. Or 99% identical sequences. In a further preferred embodiment, the mutein comprises the amino acid sequences SEQ ID NO: 31, 32 and 38.
  • mutating the glycosylation motif improves the receptor selectivity of the IL-2 protein, particularly reducing the binding of IL-2 to IL-2R ⁇ .
  • the mutant glycosylation motif that reduces the binding of IL-2 to IL-2R ⁇ is selected from: 41N-42X-43T / S; 43N-44X-45T / S; 45N-46X-47T / S; 68N -69X-70T / S; 72N-73X-74T / S, preferably the glycosylation motif 43N-44X-45T / S, wherein the amino acid positions are numbered according to SEQ ID NO: 26.
  • the mutant glycosylation motif that reduces IL-2 binding to IL-2R ⁇ is selected from: (i) T41N-F42-K43S; (ii) K43N-F44-Y45T; (iii) Y45N-M46 -P47S; (iv) E68N-V69-L70S; (v) L72N-A73-Q74T; more preferably, K43N-F44-Y45T.
  • the invention provides an IL-2 mutein comprising a mutant glycosylation motif compared to wild-type IL-2, wherein the mutein comprises one or more selected from the group consisting of Mutated glycosylation motif: 41N-42X-43T / S; 43N-44X-45T / S; 45N-46X-47T / S; 68N-69X-70T / S; 72N-73X-74T / S, preferably glycosyl The motif 43N-44X-45T / S, wherein the amino acid positions are numbered according to SEQ ID NO: 26, and wherein the mutein has reduced or eliminated IL-2R ⁇ binding compared to wild-type IL-2.
  • the present invention provides an IL-2 mutein comprising a mutant glycosylation motif compared to wild-type IL-2, wherein the mutein comprises one or more mutant sugars selected from Motifs: (i) T41N-F42-K43S; (ii) K43N-F44-Y45T; (iii) Y45N-M46-P47S; (iv) E68N-V69-L70S; (v) L72N-A73-Q74T; More preferably, the mutein comprises a mutant glycosylation motif K43N-F44-Y45T.
  • the mutant glycosylation motif is introduced into the IL-2 protein by a pair of mutations selected from the group consisting of T41N / K43S; K43N / Y45T; Y45N / P47S; E68N / L70S; and L72N / Q74T.
  • the present invention provides an IL-2 mutein having a mature region in the amino acid sequence with a wild-type IL-2 protein listed in one of SEQ ID NO: 26, 29, or 30 A mature region that is at least 85% or 90% identical and has a pairwise mutation selected from T41N / K43S; K43N / Y45T; Y45N / P47S; E68N / L70S; and L72N / Q74T, preferably having a pairwise mutation K43N / Y45T.
  • the mutein comprises a sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO : 33, 34, 35, 37, and 39.
  • the IL-2 mutein may further comprise: (i) selected from 35N-36X-37T / S; 38N-39X -40T / S; and 74N-75X-76T / S mutant glycosylation motif; and / or (ii) mutant K35Q.
  • the mutein has reduced or eliminated IL-2R ⁇ binding, and (e.g., when expressed in mammalian cells as an Fc fusion protein) has improved expression and / or purification characteristics .
  • the present invention provides an IL-2 mutein having a mature region having at least the mature region of the wild-type IL-2 protein listed in one of SEQ ID NO: 26, 29, or 30 in the amino acid sequence 85% or 90% identical mature regions with paired mutations selected from T41N / K43S; K43N / Y45T; Y45N / P47S; E68N / L70S; and L72N / Q74T; and selected from K35N; R38N / L40S; Q74N / K76T; or a mutation of K35Q.
  • the mutein comprises at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 45-47. the sequence of.
  • the present invention provides B'C 'loop chimeric IL-2 muteins and truncated IL-2 muteins formed by introducing mutations in the B'C' loop region of IL-2.
  • the IL-2 protein belongs to a short-chain type I cytokine family member with four alpha helix bundles (A, B, C, D) structure.
  • B'C 'loop region or “B'C' loop sequence” is used interchangeably and refers to the linking sequence between the B- and C-helix of the IL-2 protein.
  • the linker sequence is the sequence of residues at position 72 and residues at position 84 in the IL-2 polypeptide.
  • the linking sequence includes A73-R83 for a total of 11 amino acids.
  • the introduced mutation results in a mutant protein comprising a shortened B'C compared to wild-type IL-2 (preferably human IL-2, more preferably IL-2 comprising the SEQ ID NO: 26 sequence) '
  • the loop region ie, the linker sequence between amino acid residues aa72 and aa84 is shortened in length
  • the shortened loop region has a length of less than 10, 9, 8, 7, 6, or 5 amino acids, and is preferably 7 amino acids in length, in which amino acid residues are numbered according to SEQ ID NO: 26.
  • the IL-2 mutein of the invention is a B'C 'loop chimeric mutein.
  • the mutein contains substitutions to aa73 to aa83 sequences, such as short B ' C ' loop sequences from other members of the four-helix short chain cytokine family. It can be identified by superpose of crystal structure from other members of the IL family of four helix short chain cytokines, such as IL-15, IL-4, IL-21, or members of the IL family from non-human species (such as mice). Short B'C 'loop replacing wild type IL-2.
  • the sequence for substitution is a B'C 'loop sequence from interleukin IL-15 (especially human IL-15).
  • the sequence of residues 73-83 in wild-type IL-2 is replaced with the sequence SGDASIH.
  • the IL-2 mutein of the invention is a B'C 'loop truncated mutein.
  • the mutein contains truncations of aa73 to aa83 sequences, such as truncated by 1, 2, 3, or 4 amino acids from the C-terminus.
  • the truncated loop region i.e., the linking sequence between positions 72 and 84
  • the sequence A (Q / G) S (K / A) N (F / I) H preferably said truncated
  • the loop region has the sequence AQSKNFH or AGSKNFH.
  • the stability of the B'C' loop can be increased, thereby increasing the stability of IL-2 and / or its affinity for IL-2R ⁇ . Therefore, in one embodiment, the present invention provides an IL-2 mutein having increased stability and / or increased IL-2R ⁇ binding affinity relative to wild-type IL-2, said mutein comprising the aforementioned B A 'C' loop chimeric mutation or a B'C 'loop truncation mutation, in particular, an alternative loop sequence SGDASIH or a truncated loop sequence AQSKNFH or AGSKNFH located between positions 72 and 84.
  • a chimeric B'C 'loop mutation or a truncated B'C' loop mutation not only confers increased IL-2R ⁇ binding, but also promotes expression and / or purification of the IL-2 protein, especially Expression and / or purification in mammalian cell expression systems.
  • the invention provides an IL-2 mutein having enhanced IL-2R ⁇ binding and / or improved expression and / or purification properties relative to wild-type IL-2.
  • the IL-2 mutein comprises the aforementioned B'C 'loop chimeric mutation or B'C' loop truncation mutation, in particular, an alternative loop sequence SGDASIH or a truncated loop located between positions 72 and 84 Sequence AQSKNFH or AGSKNFH.
  • the present invention provides an IL-2 mutein having a mature region in the amino acid sequence identical to the wild-type IL-2 protein listed in one of SEQ ID NO: 26, 29, or 30 A mature region having at least 85% or 90% identity and comprising a linker sequence selected from amino acid positions 72 and 84 between: SGDASIH; AQSKNFH; AGSKNFH; AQSANFH; and AQSANIH.
  • the mutein comprises a sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO : 40-44, preferably SEQ ID NO: 40-42, and more preferably SEQ ID NO: 40 or 41.
  • the invention provides an IL-2 mutein comprising a combination mutation.
  • glycosylation mutations that introduce the IL-2 and IL-R ⁇ binding interface can be combined with each other or with a B'C 'loop mutation, preferably in combination with a B'C' loop mutation described herein .
  • the B'C 'loop mutations of the invention can also be combined with glycosylation mutations that introduce IL-2 and IL-R ⁇ binding interfaces, preferably in combination with the glycosylation mutations described herein.
  • improved properties selected from two or all of the following can be provided: ( i) reduced (or eliminated) IL-2R ⁇ binding; (ii) enhanced IL-2R ⁇ binding, and (ii) improved expression levels and purification.
  • the invention provides an IL-2 mutein, which is compared to wild-type IL-2 (preferably human IL-2, more preferably IL-2 comprising the sequence of SEQ ID NO: 26)
  • the mutant protein comprises a combination mutation: (i) selected from 41N-42X-43T / S; 43N-44X-45T / S; 45N-46X-47T / S; 68N-69X-70T / S; 72N-73X- 74T / S mutant glycosylation motif; and (ii) shortening B between amino acid positions aa72 to aa84 selected from SGDASIH and A (Q / G) S (K / A) N (F / I) H
  • SGDASIH amino acid positions aa72 to aa84 selected from SGDASIH and A (Q / G) S (K / A) N (F / I) H
  • SEQ ID NO: 26 The sequence of the 'C' loop region, in which the
  • the present invention provides an IL-2 mutein having a mature region in the amino acid sequence that is the wild-type IL-2 protein listed in one of SEQ ID NO: 26, 29, or 30 A mature region having at least 85% or 90% identity and comprising a linker sequence selected from amino acid positions 72 and 84 between: SGDASIH; AQSKNFH; AGSKNFH; AQSANFH; and AQSANIH; and have paired mutations selected from : T41N / K43S; K43N / Y45T; Y45N / P47S; E68N / L70S; and L72N / Q74T.
  • the present invention provides an IL-2 mutein having a mature region in the amino acid sequence identical to the wild-type IL-2 protein listed in one of SEQ ID NO: 26, 29, or 30 A mature region that is at least 85% or 90% identical and contains a linker sequence selected from amino acid positions 72 and 84 between: SGDASIH; AQSKNFH; or AGSKNFH; and has pairwise mutations: K43N / Y45T.
  • the mutein comprises a sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO : 48, 49 or 50, preferably SEQ ID NO: 48 or 49.
  • the mutein consists of SEQ ID NO: 48, 49, or 50.
  • the combination mutation results in IL-2 having a reduced bias to preferentially stimulate p-STATA5 signaling in CD25 + T cells, and has an enhanced ability to stimulate signaling in CD25 - T cells. Therefore, in one embodiment, the invention also provides an IL-2 mutein comprising a combination mutation:
  • the mutant protein has a reduced bias to preferentially stimulate p-STATA5 signaling in CD25 + T cells, and has an enhanced ability to stimulate signaling in CD25 - T cells.
  • the mutein comprises the sequence of SEQ ID NO: 48 or 49, or a sequence having at least 95%, 96%, or higher identity with it. More preferably, the mutein consists of the sequence of SEQ ID NO: 48 or 49.
  • the IL-2 mutein of the present invention may also have one or more mutations in other regions or positions as long as it retains the one or more beneficial properties of the IL-2 muteins of the present invention Just fine.
  • the IL-2 muteins of the invention may also contain a substitution at position 125, such as C125S, C125A, C125T, or C125V, to provide additional advantages such as improved expression or homogeneity or stability (see, for example, the United States (Patent No. 4,518,584).
  • Those skilled in the art know how to determine additional mutations that can be incorporated into the IL-2 muteins of the invention.
  • the sequence difference between the IL-2 mutein and the wild-type protein can be expressed by sequence identity, or it can be expressed by the number of different amino acids between the two.
  • the IL-2 mutein has at least 85%, 86%, 87%, 88%, 89% identity, preferably 90% or more identity, and preferably 95% identity, but preferably not more than 85% More than 97%, more preferably no more than 96% identity.
  • the IL-2 mutein and the wild-type protein may have no more than 15 between them, For example, 1-10, or 1-5 mutations. In one embodiment, the remaining mutations may be conservative substitutions.
  • the invention also provides a fusion protein comprising the IL-2 mutein of the invention.
  • the IL-mutein of the invention is fused to another polypeptide that can confer improved pharmacokinetic properties, such as albumin, more preferably an antibody Fc fragment.
  • the Fc fragment comprises a mutation that reduces or removes effector function, such as a L234A / L235A mutation or L234A / L235E / G237A that reduces binding to the Fc [gamma] receptor.
  • the Fc-containing fusion protein has an increased serum half-life.
  • the Fc-containing fusion protein also has reduced effector functions mediated by the Fc region, such as reduced or eliminated ADCC or ADCP or CDC effector functions.
  • the invention also provides an IL-2 mutein-Fc fusion protein, wherein the Fc fragment comprises an effector function, such as ADCC.
  • an effector function such as ADCC.
  • wild-type IL-2 can deplete Treg cells through fusion with Fc, through Fc-mediated (especially by binding to Fc ⁇ R) immune effector functions To improve the treatment of tumors. Therefore, the fusion of the IL-2 mutein of the present invention with improved expression and / or purification and other production properties with an Fc fragment retaining the function of an immune effector is also considered in the present invention.
  • the fusion protein comprises a mutation K35N or K35Q or a pair mutation R38N / L40S or Q74N / K76T.
  • the fusion protein comprises a substitution sequence SGDASIH or a truncated sequence A (Q / G) S (K / A) N (F / I) H between amino acid positions aa72 to aa84.
  • the fusion protein comprises 90% -99% identity with the amino acid sequence SEQ ID NO: 7, 8, 14, 20-22.
  • the fusion protein comprises no more than 0-10 or 0-5 amino acid mutations with the amino acid sequence SEQ ID NO: 12.
  • the IL-2 mutein is fused to the Fc via a linker.
  • a linker can be selected to increase the activation of CD25 - T cells by the Fc fusion protein.
  • the linker is GSGS, more preferably 2x (G4S).
  • the Fc fusion protein comprises at least 85%, at least 95%, or at least 96% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-13 and 16-25. In some embodiments, the Fc fusion protein consists of the sequences of SEQ ID NOs: 3-13 and 16-25.
  • the invention also provides immunoconjugates comprising an IL2 mutein of the invention and an antigen binding molecule.
  • the antigen-binding molecules are immunoglobulin molecules, especially IgG molecules, or antibodies or antibody fragments, especially Fab molecules and scFv molecules.
  • the antigen-binding molecule specifically binds an antigen presented on tumor cells or in the tumor environment, such as an antigen selected from the group consisting of fibroblast activating protein (FAP), A1 domain of tenascin C (TNC A1), A2 domain of tenascin C (TNC A2), extra domain B (EDB) of fibronectin, carcinoembryonic antigen (CEA), melanoma-related chondroitin sulfate proteoglycan (MCSP ).
  • FAP fibroblast activating protein
  • TNC A1 domain of tenascin C TCC A1
  • A2 domain of tenascin C TNC A2
  • EDB extra domain B
  • CEA carcinoembryonic antigen
  • MCSP melanoma-related chondroitin sulfate proteoglycan
  • the IL-2 mutein of the present invention may be connected to another molecule or an antigen-binding molecule directly or through a linker, and in some embodiments, a proteolysis is included between the two. Cutting site.
  • the invention provides a nucleic acid encoding any of the IL-2 muteins or fusions or conjugates above.
  • the polynucleotide sequence encoding the mutein of the present invention can be generated by methods known in the art, either by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence encoding wild-type IL-2.
  • the polynucleotides and nucleic acids of the present invention may contain a segment encoding a secretory signal peptide and be operably linked to a segment encoding a mutein of the present invention, so as to guide the secreted expression of the mutein of the present invention.
  • the invention also provides a vector comprising a nucleic acid of the invention.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YAC).
  • YAC yeast artificial chromosomes
  • the expression vector of the present invention is a pYDO_017 expression vector.
  • the invention also provides a host cell comprising the nucleic acid or the vector.
  • Host cells suitable for replication and supporting expression of mutant IL-2 proteins or fusions or immunoconjugates are well known in the art. Such cells can be transfected or transduced with specific expression vectors, and a large number of vector-containing cells can be grown for inoculation of large-scale fermentation tanks to obtain sufficient quantities of IL-2 mutants or fusions or immunoconjugates For clinical applications.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells).
  • polypeptides can be produced in bacteria, especially when glycosylation is not required.
  • the polypeptide can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding polypeptides, including fungal and yeast strains whose glycosylation pathways have been "humanized”, resulting in the production of Or a fully human glycosylation pattern polypeptide. See Gerngross, Nat Biotech 22, 1409-1414 (2004) and Li et al., Nat Biotech 24, 210-215 (2006).
  • Examples of useful mammalian host cell lines are monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293 or 293T cells, as described for example in Graham et al., JGen Virol 36, 59 (1977)), young Rat kidney cells (BHK), mouse sertoli cells (TM4 cells, as described, for example, in Mather, BiolReprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells ( VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2), mouse breast tumor cells (MMT060562 ), TRI cells (as described, for example, in Mather et al., Annals N.Y.
  • the host cell is a eukaryotic cell, preferably a mammalian cell such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphocyte (eg, a Y0, NSO, Sp20 cell).
  • CHO Chinese hamster ovary
  • HEK human embryonic kidney
  • a lymphocyte eg, a Y0, NSO, Sp20 cell
  • the present invention provides a method for preparing an IL-2 mutein or fusion or conjugate of the present invention, wherein the method comprises, under conditions suitable for expression of the IL-2 mutein or fusion or conjugate, Culturing a host cell comprising a nucleic acid encoding said protein or fusion or conjugate, as provided above, and optionally recovering said protein or fusion or conjugate from said host cell (or host cell culture medium) ⁇ The compound.
  • the IL-2 muteins provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art.
  • the IL-2 mutein of the present invention can be tested for its binding activity to the IL-2 receptor.
  • binding to human IL-2R ⁇ or ⁇ protein can be determined by methods known in the art, such as ELISA, Western blot, etc., or exemplary methods disclosed in the examples herein.
  • assays can be performed using flow cytometry in which cells, such as yeast display cells, transfected to express a mutant protein on the cell surface are reacted with a labeled (eg, biotin-labeled) IL-2R ⁇ or ⁇ protein.
  • mutant proteins bind to the receptor, including binding kinetics (e.g., K D value), using recombinant mutein -Fc fusion, in a biological interference layer (BLI) assay.
  • binding kinetics e.g., K D value
  • recombinant mutein -Fc fusion e.g., K D value
  • BLI biological interference layer
  • a BLI assay is used as described in the examples.
  • the signaling and / or immune activation effects that occur downstream of receptor binding can be determined.
  • assays are provided for identifying biologically active mutant IL-2 proteins.
  • Biological activity may include, for example, the ability to induce proliferation of T and / or NK cells and / or Treg cells with IL-2 receptors, to induce T and / or NK cells and / or Treg cells with IL-2 receptors.
  • the invention also provides mutant IL-2 proteins having such biological activity in vivo and / or in vitro.
  • a suitable assay for testing the ability of the IL-2 mutein of the invention to stimulate NK cells to produce IFN- ⁇ may include the steps of: culturing cultured NK cells with the mutant IL-2 protein of the invention or fusion or immunoconjugate The mixture was incubated, and then the IFN- ⁇ concentration in the medium was measured by ELISA.
  • IL-2 signaling induces several signaling pathways and is involved in JAK (Janus kinase) and STAT (signal transducers and activators of transcription) signaling molecules.
  • IL-2 The interaction of IL-2 with the receptor ⁇ and ⁇ subunits results in phosphorylation of the receptor and JAK1 and JAK3, which bind to the ⁇ and ⁇ subunits, respectively.
  • STAT5 then binds to the phosphorylated receptor and is itself phosphorylated on very important tyrosine residues. This leads to dissociation of STAT5 from the receptor, dimerization of STAT5, and translocation of STAT5 dimers to the nucleus, where they promote the transcription of target genes.
  • the ability of a mutant IL-2 polypeptide to induce signaling through the IL-2 receptor can be assessed, for example, by measuring phosphorylation of STAT5. Details of this method have been disclosed in the examples. For example, PBMCs can be treated with a mutant IL-2 polypeptide or fusion or immunoconjugate of the invention and the level of phosphorylated STAT5 can be determined by flow cytometry.
  • T cells or NK cells isolated from blood can be measured by incubating T cells or NK cells with the mutant IL-2 polypeptide or immunoconjugate of the present invention, and then measuring the ATP content in the lysates of the treated cells.
  • NK cells respond to IL-2 proliferation.
  • T cells can be pre-stimulated with lectin (PHA-M) before treatment.
  • PHA-M lectin
  • This assay allows sensitive quantification of the number of viable cells, and a number of suitable alternative assays are also known in the art (e.g. [3H] -thymidine incorporation assay, cell titration GloATP assay, AlamarBlue assay, WST- 1 measurement method, MTT measurement method).
  • mutated IL-2 on tumor growth and survival can be evaluated in a variety of animal tumor models known in the art.
  • a xenograft of a human cancer cell line can be implanted into an immunodeficient mouse and treated with a mutant IL-2 polypeptide or fusion or immunoconjugate of the invention.
  • the mutant IL of the present invention can be determined based on mortality, lifetime observations (visible symptoms of adverse effects, such as behavior, weight, body temperature), and clinical and anatomical pathology (such as measurement of blood chemical values and / or histopathological analysis).
  • vascular permeability induced by treatment with IL-2 can be examined with a vascular leak reporter in a pre-treated vascular permeability animal model.
  • the vascular leak reporter is large enough to reveal the permeability of the wild-type form of IL-2 for pretreatment.
  • the presence, absence, or extent of glycosylation can also be determined by any method known to those skilled in the art, including half-shifted molecular weight (MW) Qualitative measurements, as observed by Western blotting or from Coomassie-stained SDS-PAGE gels, while quantitative measurements may include the use of mass spectrometer technology and observation of added MW offsets corresponding to asparagine-linked glycosylation, or Mass shifts accompanied by removal of asparagine-linked glycosylation by enzymes such as peptide-N-glucosidase F (PNGase-F; SigmaAldrich, St. Louis, MO) were observed.
  • MW molecular weight
  • the present invention provides a method for obtaining an IL-2 mutein having improved properties.
  • the present invention provides a method for obtaining an IL-2 mutein, comprising the steps of:
  • the glycosylation motif is introduced in the following region selected from IL-2: aa35-40, aa41-47, aa62-64, aa68-74, aa74-76;
  • N-Making the engineered IL-2 mutein for example, in the form of an Fc fusion (eg, an FcLALA fusion), expressed in mammalian cells (eg, HEK293 or CHO cells).
  • Fc fusion eg, an FcLALA fusion
  • mammalian cells eg, HEK293 or CHO cells.
  • N-glycosylation prediction tools can be used to select sites that can be mutated to promote potential N-linked glycosylation, for example by identifying sites that can be mutated to form standard NxT / Residues at the S glycosylation site (where N is asparagine and x is any amino acid except proline).
  • the introduced glycosylation motif mutation is selected from: K35N-L36-T37; R38N-M39-L40S; T41N-F42-K43S; K43N-F44-Y45T; Y45N-M46-P47S; E62N- L63-K64T; E68N-V69-L70S; L72N-A73-Q74T; Q74N-S75-K76T.
  • the present invention provides a method for obtaining an IL-2 mutein, including the following steps:
  • the B'C'loop loop region (aa73-83) of IL-2 to form a shortened loop region, preferably replacing it with other members of the four-helix short-chain cytokine family such as IL15's B 'C'loop loop sequence to form a B'C'loop chimera, or to truncate the B'C'loop of IL-2 to form a B'C'loop truncation, preferably the shortened loop region has less than 10, 9,8, and preferably equal to 7 amino acids in length; preferably truncated by 1, 2, 3 or 4 amino acids from the C-terminus of the loop region; preferably the shortened loop region has the sequence A (Q / G) S (K / A ) N (F / I) H, or SGDASIH;
  • the engineered IL-2 mutein for example, in the form of an Fc fusion (eg, an FcLALA fusion), expressed in mammalian cells (eg, HEK293 or CHO cells).
  • Fc fusion eg, an FcLALA fusion
  • mammalian cells eg, HEK293 or CHO cells.
  • the method further comprises identifying, after protein expression and purification, improved drugability (eg, expression level and / or product stability and / or homogeneity, such as one-step Fc affinity chromatography purity).
  • improved drugability eg, expression level and / or product stability and / or homogeneity, such as one-step Fc affinity chromatography purity.
  • IL2 mutein eg., IL2 mutein.
  • a glycosylation motif mutation is introduced in the region aa35-40 or aa74-76 of the IL-2 to improve the drugability of the mutant protein.
  • the introduced glycosylation motif mutation is selected from the group consisting of: K35N-L36-T37; R38N-M39-L40S; and Q74N-S75-K76T.
  • the drugability of the mutant protein is improved by replacing the B'C'loop loop with a shortened loop such as the loop sequence of IL15 or by truncating the B'C'loop loop.
  • the shortened loop sequence is selected from: A (Q / G) S (K / A) NFH, or SGDASIH.
  • the method includes, in addition to the glycosylation mutation, introducing other point mutations, such as K35Q, to improve the drugability of the mutant protein. As will be apparent to those skilled in the art, these mutations can be combined with mutations that confer other improved properties to obtain IL-2 muteins with multiple improved properties.
  • the method further comprises identifying an IL-2 mutein that exhibits reduced (preferably eliminated) IL-2Ra binding capacity relative to wild-type IL-2.
  • the binding capacity of the IL-2 mutein to IL-2Ra is determined by measuring the affinity KD value, such as by biofilm thin-layer interference techniques.
  • the binding capacity is determined by measuring the activation efficacy of IL-2 muteins on CD25 + T cells.
  • the IL-2 mutein exhibits reduced CD25 + T cell activation efficacy relative to wild-type IL-2, as determined, for example, by measuring activation of p-STAT5 signaling in the cell.
  • the mutation is preferably introduced into the region of IL-2: aa41-47 or aa68-70 or aa72-74 to form a potential N-linked glycosylation site, and then it is tested whether the mutation results in reduced or eliminated IL-2 and IL- 2R ⁇ binding.
  • the introduced glycosylation motif mutation is selected from: T41N-F42-K43S; K43N-F44-Y45T; Y45N-M46-P47S; E68N-V69-L70S; L72N-A73-Q74T.
  • these glycosylation mutations can be combined with mutations that confer other improved properties to obtain IL-2 muteins with multiple improved properties.
  • the method further comprises identifying an IL-2 mutein that exhibits enhanced IL-2R ⁇ binding relative to wild-type IL-2.
  • the binding capacity of the IL-2 mutein to IL-2R ⁇ is determined by measuring the affinity KD value, such as by biofilm thin-layer interference techniques.
  • the binding capacity is determined by measuring the activation efficacy of IL-2 muteins on CD25 - T cells.
  • the IL-2 mutein exhibits enhanced CD25 - T cell activation efficacy relative to wild-type IL-2, as determined, for example, by measuring activation of p-STAT5 signals in the cell.
  • the B'C'loop loop is replaced with a shortened loop such as the loop sequence of IL15 or by truncating the B'C'loop loop to enhance binding to IL-2R ⁇ .
  • the shortened loop sequence is selected from: A (Q / G) S (K / A) NFH, or SGDASIH.
  • the method comprises combining the introduction of a mutation that improves drugability, a mutation that reduces IL2Ra binding, and / or a mutation that enhances IL2R ⁇ binding, and / or a combination of mutations that confer other improved properties to obtain multiple Improved IL-2 mutant protein.
  • glycosylation mutations are introduced in combination, such as truncation and / or substitution mutations in the regions aa41-47 and aa68-74, and shortening the length of the B'C'loop loop region.
  • the method comprises identifying an IL-2 mutein that exhibits reduced IL-2Ra binding and enhanced IL-2R ⁇ binding relative to wild-type IL-2, optionally identifying also having improved drugability (Eg, improved expression and / or purity, and / or product stability and / or homogeneity).
  • the parent wild-type IL-2 protein used as a mutant template preferably has at least 85%, or at least 90% or 95% identity to SEQ ID NO: 26, more preferably a human-derived IL- 2 protein.
  • the present invention also includes a composition (including a pharmaceutical composition or a pharmaceutical preparation) comprising an IL-2 mutein or a fusion or an immunoconjugate thereof, and a polynuclear core comprising an IL-2 mutein or a fusion or an immunoconjugate thereof.
  • compositions may also optionally contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, as known in the art.
  • Pharmaceutically acceptable carriers suitable for the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • water is the preferred carrier.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk , Glycerin, propylene, glycol, water, ethanol, etc.
  • compositions may also contain small amounts of wetting or emulsifying agents, or pH buffering agents.
  • these compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.
  • Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.
  • the IL-2 muteins, fusions or immunoconjugates of the invention having the desired purity can be obtained by combining one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. (ed. (1980))) to prepare a pharmaceutical formulation comprising the invention, preferably in the form of a lyophilized formulation or an aqueous solution.
  • a pharmaceutical formulation comprising the invention, preferably in the form of a lyophilized formulation or an aqueous solution.
  • An exemplary lyophilized antibody formulation is described in US Patent No. 6,267,958.
  • Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including histidine-acetate buffers.
  • sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the protein, which matrices are in the form of shaped
  • the pharmaceutical composition or formulation of the present invention may also contain one or more other active ingredients that are required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other .
  • active ingredients such as chemotherapeutic agents, PD-1 axis binding antagonists (such as anti-PD-1 antibodies or anti-PD-L1 antibodies or anti-PD-L2 antibodies).
  • the active ingredients are suitably present in combination in an amount effective for the intended use.
  • the composition further comprises a second therapeutic agent.
  • the second therapeutic agent may be an immune checkpoint inhibitor.
  • the second therapeutic agent may be selected from, including but not limited to, for example, anti-CTLA-4 antibodies, anti-CD47 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CD40 antibodies, anti-OX40 (also known as CD134, TNFRSF4, ACT35 and / or TXGP1L) antibody, anti-LAG-3 antibody, anti-CD73 antibody, anti-CD137 antibody, anti-CD27 antibody, anti-CSF-1R antibody, TLR agonist or IDO or TGF ⁇ One or more small molecule antagonists.
  • the second therapeutic agent is a PD-1 antagonist, especially an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3, an anti-CD47.
  • the second therapeutic agent may also be other radiotherapy or chemotherapy drugs.
  • the invention also provides a combination product comprising a mutein of the invention or a fusion or immunoconjugate thereof, and one or more other therapeutic agents (e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, Vaccines, anti-infective agents, etc.).
  • therapeutic agents e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, Vaccines, anti-infective agents, etc.
  • the combination product of the present invention can be used in the treatment method of the present invention.
  • the present invention provides a combination product, wherein the other therapeutic agent is, for example, a therapeutic agent, such as an antibody, effective to stimulate an immune response to further enhance, stimulate or up-regulate the immune response in a subject.
  • the other antibodies are, for example, anti-PD-1 antibodies or anti-PD-L1 antibodies or anti-PD-L2 antibodies or anti-LAG-3 antibodies or anti-CTLA-4 antibodies or anti-TIM-3 antibodies.
  • the combination product is used to prevent or treat a tumor.
  • the tumor is a cancer, such as a gastrointestinal cancer, such as gastric cancer, rectal cancer, colon cancer, colorectal cancer, etc .; or a skin cancer such as melanoma; or renal cell carcinoma, bladder cancer, non-small cell Lung cancer etc.
  • the combination product is used to prevent or treat infections, such as bacterial infections, viral infections, fungal infections, protozoan infections, and the like.
  • mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits and rodents (e.g., mice and rats mouse).
  • domesticated animals e.g., cows, sheep, cats, dogs, and horses
  • primates e.g., human and non-human primates such as monkeys
  • rabbits and rodents e.g., mice and rats mouse.
  • the subject is a human.
  • treatment refers to a clinical intervention intended to alter the natural process of a disease in an individual being treated. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, reducing symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the state of the disease, and alleviating or improving the prognosis.
  • the invention provides a method of stimulating the immune system of a subject, the method comprising administering to the subject an effective amount of a pharmaceutical combination comprising an IL-2 mutein or fusion or immunoconjugate of the invention Thing.
  • the IL-2 mutein of the present invention has high activity and selectivity for CD25 - CD122 + effector cells (cytotoxic CD8 + T cells and NK cells), and has a reduced stimulating effect on CD25 + Treg cells. Therefore, the IL-2 mutein of the present invention can be used at a low dose to stimulate the immune system of a subject.
  • the invention relates to a method of enhancing an immune response in a subject, the method comprising administering to the subject an effective amount of any of the IL-2 muteins described herein, or Its fusions or immunoconjugates.
  • an IL-2 mutein of the invention, or a fusion or immunoconjugate thereof is administered to a tumor-bearing subject to stimulate an anti-tumor immune response.
  • an antibody of the invention or an antigen-binding portion thereof is administered to a subject carrying an infection to stimulate an anti-infective immune response.
  • the IL-2 muteins of the invention can be used in combination with a Treg depleting antibody (eg, Fc ⁇ R-mediated Treg depletion) to further reduce the immunosuppressive effects caused by Treg.
  • a Treg depleting antibody eg, Fc ⁇ R-mediated Treg depletion
  • the IL-2 muteins of the invention can be administered in combination with an immune checkpoint inhibitor to, for example, enhance the effect of cancer immunotherapy, such as in combination with anti-PD-1 and anti-CTLA-4.
  • the invention in another aspect, relates to a method of treating a subject's diseases, such as tumors and cancers and infections, the method comprising administering to the subject an effective amount of any of the IL-2 muteins described herein, or Its fusions or immunoconjugates.
  • Cancer can be early, intermediate, or advanced or metastatic.
  • the tumor or tumor cell may be selected from colorectal tumor, ovarian tumor, pancreatic tumor, lung tumor, lung tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, gastrointestinal tumor, melanoma, cervical Tumors, bladder tumors, glioblastomas, and head and neck tumors.
  • the cancer can be selected from colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glia Cell tumor and head and neck cancer.
  • the tumor is melanoma, renal cell carcinoma, colorectal cancer, bladder cancer, non-small cell lung cancer.
  • the invention in another aspect, relates to a method of treating an infectious disease, such as a chronic infection, in a subject, the method comprising administering to the subject an effective amount of any of the IL-2 muteins or fragments thereof described herein Or an immunoconjugate, a multispecific antibody, or a pharmaceutical composition comprising the antibody or fragment.
  • the infection is a viral infection.
  • the method of the invention further comprises administering to the subject one or more therapies (e.g., treatment modality and / Or other therapeutic agents).
  • the treatment modality includes surgical treatment and / or radiation therapy.
  • the methods of the invention further comprise administering at least one other immunostimulatory antibody, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CD43 antibody, and / or CTLA-4 antibodies.
  • these antibodies can be, for example, fully human, chimeric, or humanized antibodies.
  • the anti-PD-1 antibody is selected from the group consisting of: IBI308 (sintilizumab, WO2017 / 025016A1), MDX-1106 (nivolumab, OPDIVO), Merck 3475 (MK-3475, pembrolizumab, KEYTRUDA), and CT-011 (Pidilizumab).
  • the anti-PD-1 antibody is MDX-1106.
  • the anti-PD-1 antibody is nivolumab (CAS registration number: 946414-94-4).
  • the IL-2 mutein or fragment thereof, alone or in combination with a PD-1 antagonist can also be administered in combination with one or more other therapies such as a treatment modality and / or other therapeutic agents.
  • the treatment modalities include surgery (e.g., tumor resection); radiation therapy (e.g., exoparticle beam therapy, which involves three-dimensional conformal radiation therapy in which the illuminated area is designed), local irradiation (e.g., pointing at a preselected target) Or organ irradiation) or focused irradiation.
  • a disease eg, a tumor
  • methods of treating a disease comprising administering to a subject a mutein and a CTLA-4 antagonist antibody described herein.
  • the anti-CTLA-4 antibody may be, for example, an antibody selected from: (ipilimumab or antibody 10D1, described in PCT Publication No. WO 01/14424), tremelimumab (formerly known as ticilimumab, CP-675,206), and anti-CTLA-4 antibodies described in the following publications: WO 98/42752; WO 00 / 37504; U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Proc. Natl. Acad. Sci.
  • a disease eg, a tumor
  • methods of treating a disease comprising administering to a subject an anti-mutant protein and an anti-LAG-3 antagonist antibody described herein.
  • the anti-LAG3 antibody may be, for example, an antibody selected from antibodies 25F7, 26H10, 25E3, 8B7, 11F2, or 17E5 described in US Patent Application Nos. US2011 / 0150892 and WO2014 / 008218, or a CDR or variable region comprising these antibodies Antibodies; BMS-986016; IMP731 described in US 2011/007023.
  • the IL-2 muteins of the invention can be administered in combination with a chemotherapeutic or chemotherapeutic agent. In some embodiments, the IL-2 muteins of the invention can be administered in combination with radiotherapy or a radiotherapy agent. In some embodiments, the IL-2 muteins of the invention can be administered in combination with a targeted therapy or a targeted therapeutic agent. In some embodiments, the IL-2 muteins of the invention can be administered in combination with an immunotherapy or immunotherapeutic agent, such as a monoclonal antibody.
  • the muteins of the present invention can be administered by any suitable method, including parenteral administration, intrapulmonary administration Drugs and intranasal administration, and if needed for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Depending on whether the medication is short-term or long-term, it can be administered by any suitable route, such as by injection, such as intravenous or subcutaneous injection.
  • Various dosing schedules are covered herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • the appropriate dosage of the mutein of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and progress of the disease , Administration for prophylactic or therapeutic purposes, previous treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician.
  • the antibody is suitably administered to a patient in one treatment or after a series of treatments.
  • the present invention also provides the use of the IL-2 mutein, composition, immunoconjugate, and fusion of the present invention in the manufacture of a medicament for the aforementioned method (for example, for treatment).
  • IL-2 glycosylation site design find out the distance between IL-2 and IL-2Ra is And the amino acid whose side chain is exposed to the solution is mutated to asparagine, and the third amino acid is mutated to serine or threonine to form an NXS / T motif (X can be any amino acid except P ), See Table 1.
  • B'C'loop the linking sequence of Bhelix and Chelix of IL-2 (Fig. 2A), including A73-R83 for a total of 11 amino acids.
  • Example 2 Expression and purification of IL-2 mutant-Fc fusion protein and IL-2 receptor
  • Wild-type IL-2 (uniprot: P60568, aa21-153, C125S, referred to as IL-2 WT), and the IL-2 mutant IL-2 3X (R38D, K43E , E61R), IL-2 glycans and B'C ' Loop chimeras and truncates were linked to human IgG1 Fc (L234A, L235A, abbreviated FcLALA, SEQ ID NO: 28) via a GSGS linker sequence, and constructed on the pTT5 vector to express the following proteins:
  • Protein name structure SEQ ID NOs Y001 IL-2 WT -GSGS-FcLALA SEQ ID NO: 1 Y002 IL-2 .3X -GSGS-FcLALA SEQ ID NO: 2
  • IL-2 WT IL-2 WT , IL-2 3X and L011 (IL-2 glycan5 ) are linked to FcLALA through two GGGGS and constructed on the vector of pCDNA3.1 to express the following proteins:
  • Protein name structure SEQ ID NOs Y038 IL-2 .glycan5 -2 * (G4S) -FcLALA SEQ ID NO: 13 Y040 IL-2 .3X -2 * (G4S) -FcLALA SEQ ID NO: 14 Y045 IL-2 WT -2 * (G4S) -FcLALA SEQ ID NO: 15
  • K35Q mutation is designed based on the aforementioned mutant protein Y007 and protein 3D structure), and is connected to FcLALA through the GSGS linker sequence And constructed on the pTT5 vector; used to express the following proteins:
  • Protein name structure SEQ ID NOs Y048 IL-2 glycan5.
  • glycan8 -GSGS-FcLALA SEQ ID NO: 16 Y049 IL-2 glycan5.
  • glycan1 -GSGS-FcLALA SEQ ID NO: 17 Y050 IL-2 glycan5.
  • K35Q -GSGS-FcLALA SEQ ID NO: 18
  • Protein name structure SEQ ID NOs Y056 IL-2 .glycan5.15BCL -2 * (G4S) -FcLALA SEQ ID NO: 19 Y081 IL-2 .glycan5.truncate1 -2 * (G4S) -FcLALA SEQ ID: NO: 24 Y082 IL-2 .glycan5.truncate2 -2 * (G4S) -FcLALA SEQ ID NO: 25
  • IL-2 3X is an IL-2 mutant reported in previous literature (Rodrigo Vazquez-Lombardi et al., Nature Communications, 8: 15373, DOI: 10.1038 / ncomms15373). It is the same as IL-2 WT and also contains a C125S mutation and contains mutations. R38D, K43E, E61R, the sequences of which are shown in SEQ ID NO: 27. According to reports, IL-2 3X does not bind to IL-2R ⁇ , and its binding capacity to IL-2R ⁇ remains comparable to that of wild-type IL-2.
  • Expi293 cells (Invitrogen) were passaged according to the required transfection volume, and the cell density was adjusted to 1.5 ⁇ 10 6 cells / ml one day before transfection. The cell density on the day of transfection was about 3 ⁇ 10 6 cells / ml. Take a final volume of 1/10 (v / v) Opti-MEM medium (Gibco article number: 31985-070) as a transfection buffer, add the expression plasmid constructed above, mix well, and filter with a 0.22 ⁇ m filter head for use.
  • PEI polyethyleneimine
  • the cell culture solution was centrifuged at 13,000 rpm for 20 minutes, and the supernatant was collected, and the supernatant was purified using a prepacked column, Hitrap, Mabselect, Sure (GE, 11-0034-95).
  • the operation is as follows: before purification, equilibrate the packed column with 5 column volumes of equilibration solution (20mM Tris, 150mM NaCl, pH7.2); pass the collected supernatant through the column, and then wash the packed column with 10 column volumes of equilibration solution, remove Non-specific binding protein; wash the packing with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5) and collect the eluate.
  • Tris (2M Tris) was added per 1 ml of the eluent, and it was exchanged into a PBS buffer (Gibco, Cat. No. 70011-044) using an ultrafiltration concentration tube (MILLIPORE, Cat. No .: UFC901096), and the concentration was measured. Take 100 ⁇ g of purified protein, adjust the concentration to 1 mg / mL, and use a gel filtration chromatography column SW3000 (TOSOH article number: 18675) to determine the protein purity.
  • the glycosylation mutants Y007, Y008, and Y014 significantly increased the expression and purity of the protein compared to Y001 by mutating one or two amino acids on the surface.
  • Y048, Y049, and Y050 increased the expression level from 7.77 mg / L to more than 50 mg / L (Y048 and Y049) or 40 mg / L by adding another glycosylation site or adding a K35Q mutation site on the basis of Y011.
  • L (Y050) purity increased from 31.35% to more than 80%, which made the drug-making property of the molecule significantly improved.
  • the B'C'loop chimera (Y017) and the truncated body (Y057 / 058/059) have greatly improved the expression level and purity of one-step affinity chromatography.
  • Human IL-2 receptors Uiprot: P01589, aa-217) and (Uiprot: P14784, aa27-240) are attached with an avi tag at the C-terminus of the sequence (a peptide: GLNDIFEAQKIEWHE, which can be biotinylated by BirA enzyme) and Six histidine tags (HHHHHH) were constructed on the pTT5 vector.
  • the method of plasmid transfection of 293F cells (Invitrogen) was the same as the expression method of IL-2Fc fusion protein.
  • the collected medium was centrifuged at 4500 rpm for 30 minutes, and the cells were discarded. The supernatant was filtered through a 0.22 ⁇ l filter.
  • the nickel column (5ml Historexcel, GE, 17-3712-06) used for purification was soaked with 0.1M NaOH for 2h, and then washed with 5-10 column volumes of ultrapure water to remove lye.
  • Example 3 Affinity determination of IL-2 mutant Fc fusion protein (abbreviation: IL-2 mutant- FC) and its receptor
  • Biolayer Interferometry (BLI) technology is used to determine the equilibrium dissociation constant (KD) of the IL-2 mutant- FC of the present invention that binds human IL-2R ⁇ and IL-2R ⁇ .
  • KD equilibrium dissociation constant
  • the BLI method affinity measurement was performed according to existing methods (Estep, P, et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5 (2): 270-8).
  • the instrument setting parameters are as follows: Operating steps: Baseline, Loading ⁇ 1nm, Baseline, Association, and Dissociation; the running time of each step depends on the sample binding and dissociation speed, the rotation speed is 400 rpm, and the temperature is 30 ° C. K D values were analyzed using ForteBio analysis software.
  • Protein name Affinity to IL-2R (M) Y001 1.12E-08 Y002 N.B. Y007 2.55E-09 Y008 4.23E-08 Y009 N.B. Y010 N.B. Y011 N.B. Y012 9.22E-08 Y013 N.B. Y014 1.03E-08 Y015 N.B.
  • N.B . IL-2 did not bind to the receptor
  • P.F Weak binding, poor fitting effect. ;
  • affinity data 1) Y009, Y010, Y011, Y013 and Y015 can block the binding of IL-2R (Table 4a); 2) B'C'loop chimeric molecules and truncated molecules, not only increase the molecular The amount of expression also increased the affinity of the molecule with IL-2R (Table 4b); 3) the combination of IL-2 glycosylation and B'C'loop modification Y056 and Y081, and Y045 (IL-2 WT -2 * ( G4S) -FcLALA), Y040 (IL-2 .3X -2 * (G4S) -FcLALA) and Y038 (IL-2 .glycan5 -2 * (G4S) -FcLALA), while blocking IL2R binding, Enhanced affinity to IL2R.
  • IL-2 WT and IL-2R ⁇ The affinity of IL-2 WT and IL-2R ⁇ is higher than that of IL-2R ⁇ and IL-2R ⁇ . It will preferentially bind to IL-2R ⁇ on the cell surface, and then recruit IL-2R ⁇ to release downstream p-STAT5 signals through IL-2R ⁇ to stimulate T cells and NK cell proliferation. Because IL-2R ⁇ is on the surface of Treg cells, and there is no IL-2R ⁇ on the surface of effector T cells and NK cells, normally IL-2 WT will preferentially stimulate Treg cells to proliferate and down-regulate the immune response.
  • IL-2 mutant does not bind to IL-2R ⁇ , which eliminates the preference of preferentially stimulating the proliferation of Treg cells, while stimulating the proliferation of T cells and NK cells, so that the number of effector T cells and NK cells is effectively increased, and the antitumor effect is improved.
  • each mutant by detecting the activation of p-STAT5 signal of primary human CD8 + T cells by each IL-2 mutant -FC, it is verified that each mutant removes the bias of activation of CD25 + cells, and screens for the effect on the activation of CD25 - cells. Strong mutant. Specific steps are as follows:
  • PBMC cells Allcells article number: PB005F, 100M pack
  • PB005F Allcells article number: PB005F, 100M pack
  • step 2 The cells in step 2 are CD8 + CD25 - T cells, and the cells in step 3 are CD8 + CD25 + T cells.
  • Y017 (IL-2 hyb15BCL -GSGS-FcLALA) activates CD25 - CD8 + T cells (EC017 value of Y017 is 0.9902) than Y001 (EC 50 value (10.69) was increased by 10.79 times ( Figure 5A); while the activation of CD25 + CD8 + T cells (Y017 with an EC50 value of 0.0018) was comparable to Y001 (with an EC 50 value of 0.0020) ( Figure 5B).
  • IL-2 at the interface adds a N-glycans on CD25 - CD8 + T cell activation (Y038 EC 50 value of 369.0) than the wild type IL-2 (Y045, EC 50 value (31.73), a decrease of 11.63 times, but better than the IL-2 3X (Y040) reported in the literature; and on the basis of the chimeric human IL-15 B'C'loop (Y056, EC 50 value of 8.571), The activation of CD25 - CD8 + T cells was 3.7 times higher than Y045 and 43.05 times higher than Y038 ( Figure 5C).

Abstract

提供新型白介素2(IL-2)突变蛋白。还提供包含该IL-2突变蛋白的融合蛋白、免疫缀合物，以及编码该IL-2突变蛋白的核酸、包含该核酸的载体和宿主细胞。进一步提供制备该IL-2突变蛋白的方法、包含该IL-2突变蛋白的药物组合物和该突变蛋白的治疗用途。

Description

新型白介素2及其用途 技术领域
本发明涉及新型白介素2(IL-2)突变蛋白及其用途。具体地，本发明涉及，与野生型IL-2相比，具有改善的性质，例如，改善的成药性、降低的IL-2Rα受体结合能力和/或提高的IL-2Rβ受体结合能力的IL-2突变蛋白。本发明还提供包含该IL-2突变蛋白的融合蛋白、免疫缀合物，以及编码该IL-2突变蛋白的核酸、包含该核酸的载体和宿主细胞。本发明进一步提供制备该IL-2突变蛋白的方法、包含该IL-2突变蛋白的药物组合物和该突变蛋白的治疗用途。
背景技术
白介素-2(IL-2)，也称作T细胞生长因子(TCGF)，是一种主要由活化的T细胞，尤其是CD4 +T辅助细胞产生的多能细胞因子。在真核细胞中，人IL-2(uniprot:P60568)作为153个氨基酸的前体多肽合成，在去除N端20个氨基酸后，产生成熟的分泌性IL-2。其它物种的IL-2的序列也已经公开，参见NCBI Ref Seq No.NP032392(小鼠)、NP446288(大鼠)或NP517425(黑猩猩)。
白介素2具有4个反平行的、两亲性α螺旋，此4个α螺旋形成其功能必不可少的四级结构(Smith,Science 240,1169-76(1988)；Bazan,Science257,410-413(1992))。在大多数情况下，IL-2通过三种不同受体起作用：白介素2受体α(IL-2Rα；CD25)、白介素2受体β(IL-2Rβ；CD122)和白介素2受体γ(IL-2Rγ；CD132)。IL-2Rβ和IL-2Rγ对于IL-2的信号传导至关重要，而IL-2Rα(CD25)对于信号传导不是必需的，但可以赋予IL-2对受体的高亲和力结合(Krieg等,Proc Natl Acad Sci 107,11906-11(2010))。由IL-2Rα,β,和γ联合形成的三聚体受体(IL-2αβγ)为IL-2高亲和力受体(KD约10pM)，由β和γ组成的二聚体受体(IL-2βγ)为中间亲和力受体(KD约1nM)，单独由α亚基形成的IL-2受体为低亲和力受体。
免疫细胞表达二聚体或三聚体IL-2受体。二聚体受体在细胞毒性CD8 +T细胞和天然杀伤细胞(NK)上表达，而三聚体受体主要在激活的淋巴细胞和CD4 +CD25 +FoxP3 +抑制性调节T细胞(Treg)上表达(Byman,O.和Sprent.J.Nat.Rev.Immunol.12,180-190(2012))。由于静息状态的效应T细胞和NK细胞在细胞表面上没有CD25，故对于IL-2相对不敏感。而Treg细胞在体内一贯表达最高水平的CD25，因此，正常情况下IL-2会优先刺激Treg细胞增殖。
IL-2通过与不同细胞上IL-2受体的结合，在免疫反应中介导多重作用。一方面，作为免疫系统刺激剂，IL-2可以刺激T细胞增殖和分化，诱导细胞毒性T淋巴细胞(CTL)生成，促进B细胞增殖和分化和免疫球蛋白合成，并刺激天然杀伤(NK)细胞的生产、增殖和活化，并由此已经被批准作为免疫治疗剂用于癌症和慢性病毒感染的治疗。另一方面，IL-2可以促进免疫抑制性CD4 +CD25 +调节性T细胞(即，Treg细胞)的维持(Fontenot等,Nature Immunol 6,1142-51(2005)；D’Cruz和Klein,Nature Immunol 6,1152-59(2005)；Maloy和Powrie,Nature Immunol6,1171-72(2005))，并介导活化诱导的细胞死亡(AICD)和参与针对自体抗原和肿瘤抗原的免疫耐受的建立和维持(Lenardo等人，Nature 353：858(1991))，从而在患者中引起由AICD导致的肿瘤耐受性和由激活的Treg细胞导致的免疫抑制。此外，高剂量IL-2在患者中可引起血管渗漏综合征(VLS)。已经证实，IL-2通过直接结合肺内皮细胞上的IL-2三聚体受体 (IL-2αβγ)而诱导肺水肿(Krieg等,Proc Nat Acad Sci USA107,11906-11(2010))。
为了克服与IL-2免疫治疗相关的上述问题，已经提出了通过改变IL-2对不同受体的选择性或偏好性，来降低IL-2治疗的毒性和/或提高其功效。例如，已经提出使用IL-2单克隆抗体与IL-2的复合物，通过使IL-2靶向表达CD122而非CD25的细胞，诱导对CD122 high群体的优先扩增，增强体内IL-2治疗效果(Boyman等,Science 311,1924-1927(2006))。Oliver AST等(US2018/0142037)提出在IL-2的氨基酸残基位置42,45和72上引入三重突变F42A/Y45A/L72G来降低对IL-2Rα受体的亲合力。Aron M.Levin等(Nature,Vol 484,p529-533,DOI:10.1038/nature10975)提出一种称作“superkine”的IL-2突变体IL-2 H9，该突变体包含五重突变L80F/R81D/L85V/I86V/I92F，具有增强的IL-2Rβ结合，由此提高了对CD25 -细胞的刺激作用，但仍保持了对CD25的高结合性。Rodrigo Vazquez-Lombardi等人(Nature Communications,8:15373,DOI:10.1038/ncomms15373)提出一种三重突变人IL-2突变蛋白IL-2 3X，该蛋白在氨基酸残基位置38,43和61分别具有残基突变R38D-K43E-E61R，导致该突变蛋白对IL-2Rα不结合，但是该突变蛋白激活CD25 -细胞的效应弱，对CD25 +细胞的激活偏向性依然存在。此外，Rodrigo Vazquez-Lombardi等人也提出了通过制备白介素2-Fc融合物来改善白介素的药效学性质，但是该融合蛋白的表达量低且易于形成聚集体。
鉴于IL-2在免疫调节和疾病中的作用，本领域仍然存在着开发具有改善性质的新IL-2分子的需求，特别是展现出有利生产、纯化、具有改善的药效学性质的IL-2分子。
发明概述
本发明通过提供相对于野生型IL-2具有改善的成药性质和/或改善的IL-2受体选择性/偏向性的新IL-2突变蛋白，满足了上述的需求。
因此，在一个方面，本发明提供了新的IL-2突变蛋白。在一些实施方案中，本发明的IL-2突变蛋白具有以下的一个或多个特性：
(i)改善的成药性，尤其是在哺乳动物细胞中表达时改善表达和/或纯化，
(ii)降低或消除与IL-2Rα的结合；
(iii)增强与IL-2Rβ的结合。
在一些实施方案中，本发明提供在IL-2与IL-2Rα的结合界面包含引入的突变糖基化基序的IL-2突变蛋白；在另一些实施方案中，本发明提供在IL-2的B’C’环区包含缺失和/或替代以具有缩短的环序列的IL-2突变蛋白；在再一些实施方案中，本发明提供具有突变的糖基化基序和缩短的B’C’环序列两者的IL-2突变蛋白。
此外，本发明提供了包含IL-2突变蛋白的融合蛋白和免疫缀合物，药物组合物和组合产品；编码IL-2突变蛋白的核酸，包含所述核酸的载体和宿主细胞；以及产生本发明的IL-突变蛋白、融合蛋白和免疫缀合物的方法。
再有，本发明也提供了利用本发明的IL-2突变蛋白和融合物和免疫缀合物治疗疾病的方法和刺激受试者免疫系统的方法和用途。在一些实施方案中，本发明方法在受试者中导致CD25 -效应T细胞和NK细胞的强激活和扩增。在再一些实施方案中，通过本发明方法，可以有效地减少IL-2在Treg细胞上介导的免疫下调作用。
在下面的附图和具体实施方案中进一步说明本发明。然而，这些附图和具体实施方案不应被认为限制本发明的范围，并且本领域技术人员容易想到的改变将包括在本发明的精神和所附权利要求的保护范围内。
附图说明
图1显示了IL-2与IL-2Rα的晶体结构(PDB:1Z92)(A)和IL-2糖基化修饰蛋白的结构示意图(B)。
图2显示了IL-2晶体结构(PBD:2ERJ)(A)和人与鼠的IL-2及人IL15的B’C’loop结构superpose(B)。
图3显示了IL-2Rα纯化后的样品HPLC纯度检测图谱。
图4显示了IL-2Rβ纯化后的样品HPLC纯度检测图谱。
图5显示了选择并构建的一些IL-2 mutant-FC在CD8 +CD25 -/CD25 +T细胞上激活p-STAT5的信号曲线。
图6显示了人白介素(IL-2)的成熟蛋白序列(SEQ ID NO:26)及其氨基酸残基编号，并显示了示例性的IL-2糖基化突变体和IL-2嵌合及截短B’C’环突变体。
发明详述
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
术语“和/或”应理解为意指可选项中的任一项或可选项中的任意两项或多项的组合。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”或“包括”某个突变或突变组合的IL-2突变蛋白时，也旨在涵盖仅具有所述突变或突变组合的IL-2突变蛋白。
在本文中，野生型“白介素-2”或“IL-2”是指作为引入本发明突变或突变组合的模板的亲本IL-2蛋白，优选天然存在的IL-2蛋白，例如来源于人、小鼠、大鼠、非人灵长类动物的天然IL-2蛋白，包括未经加工的(例如未去除信号肽)的形式和经加工的(例如去除了信号肽)的形式。包含信号肽的一个全长天然人IL-2序列显示于SEQ ID NO:29中，其成熟蛋白的序列显示于SEQ ID NO:30中。此外，该表述也包括天然存在的IL-2等位基因变体和剪接变体、同种型、同源物、和物种同源物。该表述也包括天然IL-2的变体，例如，所述变体可以与天然IL-2具有至少95％-99％或更高同一性或具有不超过1-10个或1-5个氨基酸突变(尤其是保守氨基酸取代)，并与天然IL-2蛋白具有基本相同的IL-2Rα结合亲合力和/或IL2Rβ结合亲合力。因此，在一些实施方案中，野生型IL-2相比于天然IL-2蛋白可以包含不影响其对IL-2受体结合的氨基酸突变，例如，在125位引入了突变C125S的天然人IL-2蛋白(uniprot:P60568) 属于本发明的野生型IL-2。一个包含C125S突变的野生型人IL-2蛋白的实例显示于SEQ ID NO:26中。在一些实施方案中，野生型IL-2序列可以与SEQ ID NO:26或29或30的氨基酸序列具有至少85％，95％，甚至至少96％,97％,98％,或99％或更高的氨基酸序列同一性。
在本文中，氨基酸突变可以是氨基酸取代、缺失、插入和添加。可以进行取代、缺失、插入和添加的任意组合来获得具有期望性质(例如降低的IL-2Rα结合亲合力)的最终突变蛋白构建体。氨基酸缺失和插入包括在多肽序列的氨基和/或羧基末端的缺失和插入。例如，可以在全长人IL-2位置1缺失丙氨酸残基。在一些实施方案中，优选的氨基酸突变是氨基酸取代。在另一些实施方案中，优选的氨基酸突变是氨基酸缺失。在一些实施方案中，在本发明描述的具体突变氨基酸位置上引入突变来获得具有改变的糖基化基序的IL-2突变蛋白。在一些实施方案中，在本发明描述的具体突变氨基酸位置上引入突变来获得具有缩短的B’C’环序列的IL-2突变蛋白。
在本发明中，当提及IL-2蛋白的氨基酸位置时，通过参考SEQ ID NO:26的野生型人IL-2蛋白(也称作IL-2 WT)氨基酸序列(如图6所示)予以确定。可以通过进行氨基酸序列比对(例如使用BLAST；可从http://blast.ncbi.nlm.nih.gov/Blast.cgi？PROGRAM＝blastp&PAGE_TYPE＝BlastSearch&LINK_LOC＝blasthome获得的Basic Local Alignment Search Tool，使用默认参数，进行比对)，鉴定在其它IL-2蛋白或多肽(包括全长序列或截短片段)上的对应氨基酸位置。因此，在本发明中，除非另有说明，否则IL-2蛋白或多肽中的氨基酸位置为根据SEQ ID NO:26编号的氨基酸位置。例如，当提及“F42”时，是指SEQ ID NO:26的第42位苯丙氨酸残基F，或经比对在其它IL-2多肽序列上的对应位置的氨基酸残基。
在本文中，在提及IL-2突变蛋白时，按照以下方式描述突变。氨基酸取代表示为[原始氨基酸残基/位置/取代的氨基酸残基]。例如，位置35的氨基酸取代为天冬酰胺(N)，可以表示为35N，如果35位的原始氨基酸残基是赖氨酸，则也可以表示为K35N。当取代的残基以X表示时，例如36X，是指在位置36的氨基酸可以被任何残基取代，如果X具有特定残基取值，则该位置由所定义的特定X残基取代。但在仅给出原始残基和位置的表示中，例如在本发明的突变糖基化基序K35N-L36-T37中的L36和T37，其是指在所述位置36和37不发生突变，即，36位和37位保留原始残基L和T。
在本文中，可以通过在比较窗内比较两条最佳比对的序列来确定“序列同一性百分比”。优选地，在参考序列(例如SEQ ID NO:26)的全长上确定序列同一性。用于比较的序列比对方法是本领域内公知的。适用于确定序列同一性百分比的算法包括例如BLAST和BLAST 2.0算法(参见Altschul等，Nuc.Acids Res.25:3389-402,1977和Altschul等J.Mol.Biol.215:403-10,1990。可通过美国国家生物技术信息中心(National Center for Biotechnology Information)公众获取(http://www.ncbi.nlm.nih.gov/)用于进行BLAST分析的软件。出于本申请的目的，同一性百分比通常用设置为缺省参数的BLAST2.0算法来确定。
如本文中使用的，术语“保守取代”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的生物学功能的氨基酸取代。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守取代。典型的保守型氨基酸取代是指将一种氨基酸取代为具有相似的化学性质(例如电荷或疏水性)的另一种氨基酸。以下六组各自包含彼此可进行典型保守取代的氨基酸：1)丙氨酸(A)、丝氨酸(S)、苏氨酸(T)；2)天冬氨酸(D)、谷氨酸(E)；3)天冬酰胺(N)、谷氨酰胺(Q)；4)精氨酸(R)、赖氨酸(K)；5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)； 和6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。例如，野生型IL-2蛋白可以相对于SEQ ID NO:26,29或30之一具有保守氨基酸取代，或仅具有保守氨基酸取代。再例如，本发明的突变IL-2蛋白可以相对于本文中具体给出的IL-2突变蛋白序列(例如SEQ ID NO:31-50之任一)具有保守氨基酸取代，或仅具有保守氨基酸取代。
“亲和力”或“结合亲和力”可以用于反映结合对子的成员之间相互作用的内在结合能力。分子X对其结合配偶体Y的亲和力可以由平衡解离常数(K D)表示，平衡解离常数是解离速率常数和结合速率常数(分别是k dis和k on)的比值。结合亲和力可以由本领域已知的常见方法测量。用于测量亲和力的一个具体方法是本文中的生物膜层干涉(BLI)技术测定。
在本文中，抗体结合分子是可以特异性结合抗原的多肽分子，例如，免疫球蛋白分子、抗体或抗体片段，例如Fab片段和scFv片段。
在本文中，抗体Fc片段是指含有至少一部分的恒定区的免疫球蛋白重链的C-端区域，并且可以包括天然序列Fc片段和变体Fc片段。在一个实施方案中，人IgG重链Fc片段从重链的Cys226或从Pro230延伸至羧基端。在另一实施方案中，Fc-片段的C-端赖氨酸(Lys447)可以存在或可以不存在。在另一些实施方案中，Fc片段可以包含突变，例如L234A/L235A突变。除非本文中另外指出，Fc片段中的氨基酸残基的编号根据EU编号系统，也称为EU索引，如Kabat，E.A.等，Sequences of Proteins of Immunological Interest，第5版，Public Health Service,National Institutes of Health，Bethesda，MD(1991)，NIH Publication 91-3242中所述。
本发明的各方面将在下面各小节中进一步详述。
1.本发明的IL-2突变蛋白
本发明在一方面提供新IL-2突变蛋白，所述突变蛋白具有改善的成药性质和/或改善的IL-2受体选择性/偏好性。
本发明IL-2突变蛋白的有利生物学性质
IL-2蛋白通过与IL-2受体相互作用来引发信号传导和发挥功能。野生型IL-2对不同IL-2受体显示出不同的亲合力。在静息效应细胞(包括CD8 +细胞毒性T细胞和NK细胞)上表达与野生型IL-2具有较低亲合力的IL-2β和γ受体。在调节性T细胞(Treg)细胞和激活的效应细胞上表达与野生型IL-2具有高亲合力的IL-2Rα。由于高亲合力的原因，野生型IL-2会优先结合细胞表面的IL-2Rα，再招募IL-2Rβγ，通过IL-2Rβγ释放下游p-STAT5信号，刺激Treg细胞和激活的效应细胞。因此，不受理论的束缚，降低或消除IL-2对IL-2Rα受体的亲合力，将降低IL-2优先激活CD25 +细胞的偏向性，降低IL-2介导的Treg细胞的免疫下调作用。不受理论的束缚，维持或增强对IL-2β受体的亲合力将保留或增强IL-2对效应细胞如CD8 +细胞毒性T细胞和NK细胞的激活作用和由此IL-2的免疫刺激作用。
本发明人发现，可以通过在IL-2与IL-2Rα受体结合界面引入一个或多个特定的N糖基化基序，改善IL-2突变蛋白的表达和/或纯度、和/或降低IL-2突变蛋白对IL-2Rα的结合。此外，本发明人还发现，可以通过将IL-2本身的B’C’环序列替换为来自其它白介素细胞因子例如IL-15的短B’C’环序列，或通过对IL-2本身的B’C’环序列进行截短，来增加IL-2的表达和/或纯度，并同时提高对IL-2Rβ的亲和力。
由此，本发明提供了具有改善的性质的IL-2突变蛋白。本发明的IL-2突变蛋白，相对于野生型IL-2，可以具有选自例如以下的一项或多项的改善性质：(i)在哺乳动物细胞中表达时改善的表达和/或纯度；(ii)降低或消除的对IL-2Rα受体的结合；和/或(iii)增强的对IL-2Rβ受体的结合。
在一些实施方案中，本发明的IL-2突变蛋白，相对于野生型IL-2，具有选自例如以下的一项或多项的改善性质：
(1)对IL-2Rα受体的结合亲合力降低或消除，
(2)对IL-2Rβ受体的结合亲合力增强；
(3)对高亲合力IL-2R受体(IL-2Rαβγ)的结合亲合力降低；
(4)对中等亲合力IL-2R受体(IL-2Rβγ)的结合亲合力增加；
(5)在CD25 +细胞(特别是激活的CD8 +T细胞和Treg细胞)中激活IL-2信号传导，尤其是激活STAT5磷酸化信号的能力降低；
(6)导致减少的由IL-2介导的CD25 +细胞(特别是激活的CD8 +T细胞和Treg细胞)激活和增殖；
(7)降低或消除IL-2优先刺激Treg细胞增殖的偏向性；
(8)降低Treg细胞在IL-2诱导下的免疫下调作用；
(9)保持或增强，尤其是增强，对CD25 -细胞(尤其是CD25 -T效应细胞和NK细胞)的激活作用；
(10)导致增加的由IL-2介导的效应T细胞与NK细胞激活和增殖；
(11)导致提高的免疫刺激作用；
(12)提高抗肿瘤效应。
在一些实施方案中，本发明IL-2突变蛋白具有上述(1)的性质，优选地进一步具有选自(3)和(5)-(8)的一项或多项，尤其是所有的性质；更优选地还进一步具有选自(2)和(9)-(12)的一项或多项，尤其是所有性质。在一些实施方案中，本发明IL-2突变蛋白具有上述(2)的性质，优选地进一步具有选自(9)-(12)的一项或多项，尤其是所有的性质；更优选地还进一步具有选自(1),(3)和(5)-(8)的一项或多项，尤其是所有的性质。
在一些优选实施方案中，本发明IL-2突变蛋白相对于野生型IL-2还具有以下一个或多个性质：减小的由IL-2与高亲合力受体IL-2αβγ结合介导的体内毒性。
在一些实施方案中，本发明IL-2突变蛋白具有改善的成药性质，例如，当在哺乳动物细胞例如H293T细胞中表达时，优选地以Fc融合蛋白表达时，具有选自以下的一项或多项性质：(i)优于野生型IL-2蛋白的表达量；(ii)优于野生型IL-2蛋白的稳定性；和(iii)易于纯化至更高的蛋白纯度。
在本发明的一些实施方案中，本发明IL-2突变蛋白与野生IL-2相比表现出表达水平的增加。在本发明的一些实施方案中，增加的表达发生在哺乳动物细胞表达系统中。表达水平可通过允许定量或半定量分析细胞培养上清液(优选一步亲和层析纯化后的上清液)中的重组IL-2蛋白量的任何合适方法来测定。例如，可以通过蛋白质印迹或ELISA，评估样品中的重组IL-2蛋白量。在一些实施方案中，本发明IL-2突变蛋白，与野生型IL-2相比，在哺乳动物细胞中的表达量增加至少1.1倍，或至少1.5倍，或至少2倍、3倍或4倍以上。
在一些实施方案中，如通过测定蛋白A亲和层析后纯化蛋白的纯度所显示的，本发明IL-2突变蛋白-Fc融合物，相对于野生型IL-2蛋白融合物，表现出更好的稳定性，例如具有更少的形成聚集体的倾向。在一些实施方案中，蛋白纯度通过SEC-HPLC技术检测。在一些优选的实施方案中，一步蛋白A亲和层析纯化后，本发明IL-2突变蛋白产物的纯度可以达到70％，或80％，或90％以上。
在一些实施方案中，相对于野生型IL-2(例如SEQ ID NO:26中所示IL-2 WT)，本发明IL-2突变蛋白对IL-2Rα受体的结合亲和力降低至少5倍、至少10倍、或至少25倍，尤其是至少30倍、50倍或100倍以上。在优选的实施方案中，本发明的突变蛋白不结合IL-2受体α。结合亲和力可以通过生物膜层干涉(BLI)技术测定本发明IL-2突变蛋白，例如与Fc片段融合的本发明IL-2突变蛋白，与受体IL-2Rα受体的平衡解离常数(K D)来确定。在一些实施方案中，通过BLI技术测定IL-2突变蛋白(例如以Fc融合物的形式)与受体IL-2Rα或IL-2Rβ的单价结合亲和力。
在一些实施方案中，相对于野生型IL-2(例如SEQ ID NO:26中所示IL-2 WT)，本发明IL-2突变蛋白对IL-2Rβ受体的结合亲和力增强至少5倍，至少10倍，或至少25倍，尤其是至少30倍、50倍或100倍，更优选地，至少150倍、200倍、250倍、300倍、350倍、400倍、450倍、或500倍或550倍以上。结合亲和力可以通过生物膜层干涉(BLI)技术测定本发明IL-2突变蛋白，例如与Fc片段融合的本发明IL-2突变蛋白，与受体IL-2Rβ受体的平衡解离常数(K D)来确定。在一个实施方案中，以IL-2-Fc融合蛋白形式，在BLI测定中(例如实施例中所述的BLI测定)，本发明IL-2突变蛋白与受体IL-2Rβ受体的单价结合亲和力K D值小于10.0E-07M，例如8.0E-07M至1.0E-07M，例如4.0E-07M,3.0E-07M，2.0E-07M,1.0E-07M,更优选地小于10.0E-08M,例如小于9.0E-10M。
在一个实施方案中，相对于野生型IL-2，本发明IL-2突变蛋白导致减少的由IL-2介导的CD25 +细胞激活和增殖。在一个实施方案中，CD25 +细胞是CD25 +CD8 +T细胞。在另一实施方案中，CD25 +细胞是Treg细胞。在一个实施方案中，在STAT5磷酸化测定试验中，通过检测IL-2突变蛋白在CD25 +细胞中对STAT5磷酸化信号的激活，来鉴定IL-2突变蛋白激活CD25 +细胞的能力。例如，如本申请实施例中所述，可以通过流式细胞术分析细胞中的STAT5磷酸化，确定半最大有效浓度(EC50)。
在一个实施方案中，相对于野生型IL-2，本发明IL-2突变蛋白导致保持的或增强的由IL-2介导的CD25 -效应细胞激活和增殖。在一个实施方案中，CD25 -细胞是CD8 +效应T细胞或NK细胞。在一个实施方案中，在STAT5磷酸化测定试验中，通过检测IL-2突变蛋白在CD25 -细胞中激活STAT5磷酸化信号的EC50值，来鉴定IL-2突变蛋白激活CD25 -细胞的能力。在一个实施方案中，如在STAT5磷酸化测定试验中测定的，本发明IL-2突变蛋白相对于野生型IL-2蛋白(例如SEQ ID NO:26的人IL-2)，激活CD25 +细胞的能力提高了至少1倍、例如2倍，3倍、4倍、5倍、6倍、7倍、8倍、9倍、或10倍。
在一个实施方案中，相对于野生型IL-2，本发明IL-2突变蛋白去除或降低IL-2对CD25 +细胞优先激活的偏向性。在一个实施方案中，CD25 +细胞是CD25 +CD8 +T细胞。在另一实施方案中，CD25 +细胞是Treg细胞。在一个实施方案中，在STAT5磷酸化测定试验中，通过检测IL-2突变蛋白分别在CD25 -细胞中和在CD25 +细胞中激活STAT5磷酸化信号的EC50值，来鉴定IL-2突变蛋白激活CD25 -细胞的能力。例如，通过计算在CD25 -和CD25 +T细胞上激 活STAT5磷酸化信号的EC50值的比值，确定IL-2突变蛋白对CD25 +细胞的激活偏向性。优选地，相对于野生型蛋白，突变蛋白对CD25 +的偏向性降低了至少10倍，优选至少100倍，150倍，或200倍。
本发明的突变蛋白
糖基化突变蛋白
在一方面，本发明提供了在IL-2和IL-2Ra结合界面包含突变糖基化基序的IL-2突变蛋白。
如本领域已知，多肽典型地经N-连接或O连接进行糖基化。N-连接糖基化指碳水化合物部分连接至天冬酰胺残基的侧链。三肽序列天冬酰胺-X-丝氨酸(N X S)及天冬酰胺-X-苏氨酸(N X T)为N-连接糖基化基序，其中X为除脯氨酸以外的任何氨基酸。这些三肽序列的任一者在多肽中的存在将导致潜在的糖基化位点。向蛋白质(例如IL-2)添加N连接的糖基化位点可以便利地通过改变氨基酸序列使得其含有一个或多个上述三肽序列来完成。例如，可通过改变用于单一氨基酸的密码子添加N-连接的糖基化位点。例如，编码N-X-z(其中z为任何氨基酸)的密码子可经改变以编码N-X-T(或N-X-S)，或编码y-X-T/S的密码子可经改变以编码N-X-T/S。可替代地，可同时改变编码两个氨基酸的密码子，以引入N-连接的糖基化位点(例如，y-X-z的密码子可经改变以编码N-X-T/S)。
在本文中，因引入的突变而出现在IL-2蛋白中的糖基化基序，可以以突变糖基化基序来描述。例如，突变糖基化基序K35N-L36-T37是由35位赖氨酸替代为天冬酰胺而36和37位残基保持不变而形成的N-连接糖基化基序。在本发明优选的实施方案中，引入的突变糖基化基序为N-连接的糖基化基序，N-X-S/T，其中X是除脯氨酸外的任何氨基酸。在一些实施方案中，例如，X可以是与野生型IL-2相应位置的氨基酸相同的氨基酸，或是其保守取代残基。
在一些实施方案中，本发明提供IL-2糖基化突变蛋白，与野生型IL-2(优选人IL-2，更优选包含SEQ ID NO:26序列的IL-2)相比，所述突变蛋白包含至少一个突变，所述突变在选自以下的氨基酸位置引入一个或多个糖基化基序N-X-S/T：
35N-36X-37T/S；38N-39X-40T/S；41N-42X-43T/S；43N-44X-45T/S；45N-46X-47T/S；62N-63X-64T/S；68N-69X-70T/S；72N-73X-74T/S；74N-75X-76T/S，其中，X是除脯氨酸外的任何氨基酸，优选地X是与野生型IL-2相应位置的氨基酸相同的氨基酸，或是其保守取代残基；其中氨基酸位置根据SEQ ID NO:26编号。在一些实施方案中，所引入的N连接糖基化位点的数量可以是一个以上，例如两个糖基化位点。不同的糖基化位点可以赋予IL-2不同的性质，例如一些糖基化位点可以赋予改善的表达和/或纯化性质，一些糖基化位点可以改善的IL-2受体选择性。在再一些实施方案中，除了通过突变引入的糖基化基序外，本发明突变蛋白还可以包含与野生型IL-2不同的至少1-30个氨基酸残基，例如1-20,1-15，1-10，或1-5个不同的氨基酸残基。这些不同残基可以是保守取代，也可以是赋予IL-2其它改善性质的其它突变。
改善成药性质的糖基化突变
在一些实施方案中，突变糖基化基序改善IL-2蛋白的成药性质，尤其是促进IL-2蛋白的 表达和/或纯化。
在一个实施方案中，所述改善成药性质的突变糖基化基序选自：35N-36X-37T/S；38N-39X-40T/S；和74N-75X-76T/S。在一个优选实施方案中，突变糖基化基序选自：(i)K35N-L36-T37；(ii)R38N-M39-L40S；和(iii)Q74N-S75-K76T。在一个更优选的实施方案中，突变糖基化基序是K35N-L36-T37。
由此，在一些实施方案中，本发明提供，与野生型IL-2相比，包含突变糖基化基序的IL-2突变蛋白，所述突变糖基化基序选自：35N-36X-37T/S；38N-39X-40T/S；和74N-75X-76T/S，且所述突变蛋白具有改善的成药性质。在一个实施方案中，当在哺乳动物细胞中表达时，优选地以Fc融合蛋白形式表达IL-2突变蛋白时，所述突变可以促进突变蛋白的表达和/或纯化。在再一实施方案中，所述突变可以促进IL-2的稳定性，例如以Fc融合蛋白表达时在生产过程中相比野生型IL-2具有降低的形成聚集体的倾向。例如，在表达和一步蛋白A亲和纯化后，突变蛋白可以具有比野生型蛋白更高的纯度。在一个优选实施方案中，与野生型IL-2相比，所述突变蛋白包含选自以下的突变糖基化基序：(i)K35N-L36-T37；(ii)R38N-M39-L40S；(iii)Q74N-S75-K76T；更优选地所述突变蛋白包含突变糖基化基序K35N-L36-T37。
在一些实施方案中，突变糖基化基序通过突变K35N而引入IL-2蛋白。在一些实施方案中，本发明提供这样的IL-2突变蛋白，其具有在氨基酸序列上与SEQ ID NO：26,29或30之一中所列出的野生型IL-2蛋白的成熟区具有至少90％同一性的成熟区，且具有氨基酸残基T37和突变K35N。
在一些实施方案中，突变糖基化基序通过选自R38N/L40S或Q74N/K76T的成对突变而引入IL-2蛋白。在一些实施方案中，本发明提供这样的IL-2突变蛋白，其具有在氨基酸序列上与SEQ ID NO：26,29或30之一中所列出的野生型IL-2蛋白的成熟区具有至少90％同一性的成熟区，且具有选自R38N/L40S或Q74N/K76T的成对突变。
在一些实施方案中，突变蛋白包含与选自SEQ ID NO:31，32和38的氨基酸序列具有至少90％、92％、93％、94％、95％、96％、97％、或98％或99％的同一性的序列。在再一优选的实施方案中，所述突变蛋白包含氨基酸序列SEQ ID NO:31，32和38。
降低IL-2Rα结合的糖基化突变
在一些实施方案中，突变糖基化基序改善IL-2蛋白的受体选择性，尤其是降低IL-2与IL-2Rα的结合。
在一个实施方案中，降低IL-2与IL-2Rα结合的突变糖基化基序选自：41N-42X-43T/S；43N-44X-45T/S；45N-46X-47T/S；68N-69X-70T/S；72N-73X-74T/S，优选糖基化基序43N-44X-45T/S，其中氨基酸位置根据SEQ ID NO:26编号。在一个优选实施方案中，降低IL-2与IL-2Rα结合的突变糖基化基序选自：(i)T41N-F42-K43S；(ii)K43N-F44-Y45T；(iii)Y45N-M46-P47S；(iv)E68N-V69-L70S；(v)L72N-A73-Q74T；更优选地，K43N-F44-Y45T。
由此，在一些实施方案中，本发明提供，与野生型IL-2相比，包含突变糖基化基序的IL-2突变蛋白，其中所述突变蛋白包含一个或多个选自以下的突变糖基化基序：41N-42X-43T/S；43N-44X-45T/S；45N-46X-47T/S；68N-69X-70T/S；72N-73X-74T/S，优选糖基化基序43N-44X-45T/S，其中氨基酸位置根据SEQ ID NO:26编号，且其中，与野生型IL-2相比， 所述突变蛋白具有降低的或消除的IL-2Rα结合。
在再一实施方案中，本发明提供，与野生型IL-2相比，包含突变糖基化基序的IL-2突变蛋白，其中所述突变蛋白包含一个或多个选自以下的突变糖基化基序：(i)T41N-F42-K43S；(ii)K43N-F44-Y45T；(iii)Y45N-M46-P47S；(iv)E68N-V69-L70S；(v)L72N-A73-Q74T；更优选地，所述突变蛋白包含突变糖基化基序K43N-F44-Y45T。
在一些实施方案中，突变糖基化基序通过选自T41N/K43S；K43N/Y45T；Y45N/P47S；E68N/L70S；和L72N/Q74T的成对突变，而引入IL-2蛋白中。在一些实施方案中，本发明提供这样的IL-2突变蛋白，其具有在氨基酸序列上与SEQ ID NO：26,29或30之一中所列出的野生型IL-2蛋白的成熟区具有至少85％或90％同一性的成熟区，且具有选自T41N/K43S；K43N/Y45T；Y45N/P47S；E68N/L70S；和L72N/Q74T的成对突变，优选具有成对突变K43N/Y45T。在一些实施方案中，突变蛋白包含与选自以下的氨基酸序列具有至少90％、92％、93％、94％、95％、96％、97％、或98％同一性的序列：SEQ ID NO:33,34,35,37，和39。
在一些实施方案中，除了上述降低IL-2与IL-2Rα结合的突变糖基化基序，IL-2突变蛋白还可以包含：(i)选自35N-36X-37T/S；38N-39X-40T/S；和74N-75X-76T/S的突变糖基化基序；和/或(ii)突变K35Q。与野生型IL-2相比，所述突变蛋白具有降低的或消除的IL-2Rα结合，且(例如在以Fc融合蛋白形式在哺乳动物细胞中表达时)具有改善的表达和/或纯化特性。在一些优选实施方案中，本发明提供IL-2突变蛋白，其具有在氨基酸序列上与SEQ ID NO：26,29或30之一中所列出的野生型IL-2蛋白的成熟区具有至少85％或90％同一性的成熟区，且具有选自T41N/K43S；K43N/Y45T；Y45N/P47S；E68N/L70S；和L72N/Q74T的成对突变，并具有选自K35N；R38N/L40S；Q74N/K76T；或K35Q的突变。在一些实施方案中，突变蛋白包含与选自SEQ ID NO:45-47的氨基酸序列具有至少90％、92％、93％、94％、95％、96％、97％、或98％同一性的序列。
B’C’环嵌合突变蛋白和截短突变蛋白
在一个方面，本发明提供在IL-2的B’C’环区引入突变形成的B’C’环嵌合IL-2突变蛋白和截短IL-2突变蛋白。IL-2蛋白属于具有四个α螺旋束(A,B,C,D)结构的短链I型细胞因子家族成员。在本文中，“B’C’环区”或“B’C’环序列”可互换使用，是指IL-2蛋白的B螺旋和C螺旋之间的连接序列。在一个实施方案中，根据SEQ ID NO:26的编号，该连接序列是IL-2多肽中连接位置72的残基和位置84的残基的序列。在SEQ ID NO:26、29和30的野生型蛋白中，该连接序列包括A73-R83共11个氨基酸。
在一些实施方案中，所引入的突变导致，与野生型IL-2(优选人IL-2，更优选包含SEQ ID NO:26序列的IL-2)相比，突变蛋白包含缩短的B’C’环区(即，氨基酸残基aa72和aa84之间的连接序列长度缩短)，优选地，所述缩短的环区具有小于10，9,8，7，6个或5个氨基酸长度，且优选7个氨基酸长度，其中氨基酸残基根据SEQ ID NO:26编号。
在一些实施方案中，本发明的IL-2突变蛋白为B’C’环嵌合突变蛋白。相对于野生型IL-2，所述突变蛋白包含对aa73至aa83序列的替代，例如替代为来自其它四螺旋短链细胞因子家族成员的短B’C’环序列。可以通过晶体结构的superpose，从其它四螺旋短链细胞因子IL家族成员，例如IL-15，IL-4，IL-21，或来自非人物种(如小鼠)的IL家族成员，鉴定适用于替代野生型IL-2的短B’C’环。在一个优选实施方案中，用于替代的序列为来自白介素IL-15(尤 其是人IL-15)的B’C’环序列。优选地，野生型IL-2中残基73-83的序列被替代为序列SGDASIH。
在一些实施方案中，本发明的IL-2突变蛋白为B’C’环截短突变蛋白。相对于野生型IL-2，所述突变蛋白包含对aa73至aa83序列的截短，例如自C端截短1、2、3或4个氨基酸。优选地，截短的环区(即，位置72和位置84之间的连接序列)具有序列A(Q/G)S(K/A)N(F/I)H，优选所述截短的环区具有序列AQSKNFH或AGSKNFH。
在一个实施方案中，通过对B’C’环的替代或截短，可以增加B’C’环的稳定性，从而增加IL-2的稳定性和/或与IL-2Rβ的亲和力。因此，在一个实施方案中，本发明提供，相对于野生型IL-2，具有增加的稳定性和/或增加的IL-2Rβ结合亲和力的IL-2突变蛋白，所述突变蛋白包含前述的B’C’环嵌合突变或B’C’环截短突变，尤其是，位于位置72和位置84之间的替代的环序列SGDASIH或截短的环序列AQSKNFH或AGSKNFH。
在一个实施方案中，嵌合的B’C’环突变或截短的B’C’环突变不仅赋予增加的IL-2Rβ结合，也可以促进IL-2蛋白的表达和/或纯化，尤其是在哺乳动物细胞表达系统中的表达和/或纯化。由此，在一个实施方案中，本发明提供，相对于野生型IL-2，具有增强的IL-2Rβ结合和/或改善的表达和/或纯化性质的IL-2突变蛋白。所述IL-2突变蛋白包含前述的B’C’环嵌合突变或B’C’环截短突变，尤其是，位于位置72和位置84之间的替代的环序列SGDASIH或截短的环序列AQSKNFH或AGSKNFH。
在一些优选实施方案中，本发明提供这样的IL-2突变蛋白，其具有在氨基酸序列上与SEQ ID NO：26,29或30之一中所列出的野生型IL-2蛋白的成熟区具有至少85％或90％同一性的成熟区，且在氨基酸位置72和84之间包含选自以下的连接序列：SGDASIH；AQSKNFH；AGSKNFH；AQSANFH；和AQSANIH。在一些实施方案中，突变蛋白包含与选自以下的氨基酸序列具有至少90％、92％、93％、94％、95％、96％、97％、或98％同一性的序列：SEQ ID NO:40-44，优选SEQ ID NO:40-42，更优选SEQ ID NO:40或41。
组合突变蛋白
在一方面，本发明提供包含组合突变的IL-2突变蛋白。在一个实施方案中，引入IL-2和IL-Rα结合界面的糖基化突变可以相互组合，也可以与B’C’环突变组合，优选地与本文中描述的B’C’环突变组合。在另一实施方案中，本发明B’C’环突变也可以与引入IL-2和IL-Rα结合界面的糖基化突变组合，优选地与本文中描述的糖基化突变组合。在一个优选的实施方案中，通过将B’C’环突变与引入IL-2和IL-Rα结合界面的糖基化突变组合，可以提供选自以下两项或全部三项的改善性质：(i)降低的(或消除的)IL-2Rα结合；(ii)增强的IL-2Rα结合，和(ii)改善的表达水平和纯化。
由此，在一个实施实施方案中，本发明提供IL-2突变蛋白，其中，与野生型IL-2(优选人IL-2，更优选包含SEQ ID NO:26序列的IL-2)相比，所述突变蛋白包含组合突变：(i)选自41N-42X-43T/S；43N-44X-45T/S；45N-46X-47T/S；68N-69X-70T/S；72N-73X-74T/S的突变糖基化基序；和(ii)在氨基酸位置aa72至aa84之间、选自SGDASIH和A(Q/G)S(K/A)N(F/I)H的缩短B’C’环区序列，其中氨基酸位置根据SEQ ID NO:26编号。
在一些优选实施方案中，本发明提供这样的IL-2突变蛋白，其具有在氨基酸序列上与SEQ  ID NO：26,29或30之一中所列出的野生型IL-2蛋白的成熟区具有至少85％或90％同一性的成熟区，且在氨基酸位置72和84之间包含选自以下的连接序列：SGDASIH；AQSKNFH；AGSKNFH；AQSANFH；和AQSANIH；并具有选自以下的成对突变：T41N/K43S；K43N/Y45T；Y45N/P47S；E68N/L70S；和L72N/Q74T。在一些优选实施方案中，本发明提供这样的IL-2突变蛋白，其具有在氨基酸序列上与SEQ ID NO：26,29或30之一中所列出的野生型IL-2蛋白的成熟区具有至少85％或90％同一性的成熟区，且在氨基酸位置72和84之间包含选自以下的连接序列：SGDASIH；AQSKNFH；或AGSKNFH；并具有成对突变：K43N/Y45T。在一些实施方案中，突变蛋白包含与选自以下的氨基酸序列具有至少90％、92％、93％、94％、95％、96％、97％、或98％同一性的序列：SEQ ID NO:48，49或50，优选SEQ ID NO:48或49。在一些实施方案中，所述突变蛋白由SEQ ID NO：48、49或50组成。
在一些实施方案中，组合突变导致IL-2具有降低的优先刺激CD25 +T细胞中p-STATA5信号传导的偏向性，且具有增强的刺激CD25 -T细胞中信号传导的能力。因此，在一个实施方案中，本发明也提供这样的IL-2突变蛋白，所述突变蛋白包含组合突变：
(i)在氨基酸位置43-45的突变糖基化基序K43N-F44-Y45T和在氨基酸位置aa72至aa84之间的替代序列SGDASIH；或
(ii)在氨基酸位置43-45的突变糖基化基序K43N-F44-Y45T和在氨基酸位置aa72至aa84之间的截短序列AQSKNFH，
且所述突变蛋白，与野生型IL-2相比，具有降低的优先刺激CD25 +T细胞中p-STATA5信号传导的偏向性，且具有增强的刺激CD25 -T细胞中信号传导的能力。优选地，所述突变蛋白包含SEQ ID NO:48或49的序列，或与其具有至少95％,96％、或更高的同一性的序列。更优选地，所述突变蛋白由SEQ ID NO:48或49的序列组成。
其它突变
除了上述区域和位置中的突变，本发明的IL-2突变蛋白还可以在其它区域或位置上具有一个或多个突变，只要其保留本发明IL-2突变蛋白的上述一个或多个有益性质即可。例如，本发明IL-2突变蛋白还可以包含在位置125的取代，例如C125S，C125A,C125T,或C125V，以提供额外的优点，例如改善的表达或同质性或稳定性(参见例如，美国专利号4,518,584)。本领域技术人员知晓如何确定可以并入本发明IL-2突变蛋白中的额外突变。
IL-2突变蛋白与野生型蛋白之间的序列差异性可以用序列同一性表述，也可以用两者之间差异氨基酸的数量来表达。在一个实施方案中，IL-2突变蛋白与野生型蛋白之间具有至少85％，86％,87％,88％,89％同一性，优选90％以上同一性，优选95％，但优选不超过97％，更优选不超过96％同一性。在另一实施方案中，除了本发明的上述糖基化突变或B’C’环突变或两者的组合突变外，IL-2突变蛋白与野生型蛋白之间还可以具有不超过15个，例如1-10个，或1-5个突变。在一个实施方案中，所述其余突变可以是保守取代。
2.融合蛋白和免疫缀合物
本发明还提供包含本发明IL-2突变蛋白的融合蛋白。在一个优选的实施方案中，本发明IL-突变蛋白与可以赋予改善的药代动力学性质的另一多肽融合，例如清蛋白，更优选抗体Fc 片段。在一个实施方案中，Fc片段包含减小或去除效应子功能的突变，例如降低与Fcγ受体结合的L234A/L235A突变或L234A/L235E/G237A。优选地，包含Fc的融合蛋白具有增加的血清半衰期。在一个优选的实施方案中，包含Fc的融合蛋白同时还具有减少的由Fc区介导的效应子功能，例如减少的或消除的ADCC或ADCP或CDC效应子功能。
在一个实施方案中，本发明也提供这样的IL-2突变蛋白-Fc融合蛋白，其中Fc片段包含效应子功能，例如ADCC。如文献报道的(Rodrigo Vazquez-Lombardi等，同上引文)，野生型IL-2可以通过与Fc融合，通过Fc-介导(尤其是由结合FcγR所介导)的免疫效应子功能而耗竭Treg细胞，从而改善对肿瘤治疗。因此，将具有改善的表达和/或纯化等生产性质的本发明IL-2突变蛋白，与保留免疫效应子功能的Fc片段融合，也落入本发明的考虑中。在一个实施方案中，所述融合蛋白包含突变K35N或K35Q或成对突变R38N/L40S或Q74N/K76T。在另一些实施方案中，所述融合蛋白包含在氨基酸位置aa72至aa84之间的替代序列SGDASIH或截短序列A(Q/G)S(K/A)N(F/I)H。在一个实施方案中，所述融合蛋白包含与氨基酸序列SEQ ID NO:7,8,14，20-22具有90％-99％以上同一性。在另一实施方案中，所述融合蛋白包含与氨基酸序列SEQ ID NO:12具有不超过0-10或0-5个氨基酸突变。
在一些实施方案中，IL-2突变蛋白通过接头与Fc融合。在一些实施方案中，可以选择接头以提高Fc融合蛋白对于CD25 -T细胞的激活作用。在一个实施方案中，接头是GSGS，更优选为2x(G4S)。
在一些实施方案中，Fc融合蛋白包含与选自以下的氨基酸序列至少85％，至少95％,或至少96％的同一性：SEQ ID NO:3-13和16-25。在一些实施方案中，Fc融合蛋白由SEQ ID NO:3-13和16-25的序列组成。
本发明还提供免疫缀合物，其包含本发明的IL2突变蛋白和抗原结合分子。优选地，抗原结合分子是免疫球蛋白分子，特别是IgG分子，或抗体或抗体片段，特别是Fab分子和scFv分子。在一些实施方案中，所述抗原结合分子特异性结合肿瘤细胞上或肿瘤环境中呈现的抗原，例如选自以下的抗原：成纤维细胞活化蛋白(FAP)、生腱蛋白C的A1域(TNC A1)、生腱蛋白C的A2域(TNC A2)、纤连蛋白的外域B(Extra Domain B,EDB)、癌胚抗原(CEA)、和黑素瘤有关的硫酸软骨素蛋白聚糖(MCSP)。由此，本发明免疫缀合物在施用于受试者体内后可以靶向肿瘤细胞或肿瘤环境，从而提供进一步的治疗益处，例如以更低的剂量进行治疗的可行性和由此带来的低副作用；增强的抗肿瘤效应等。
在本发明的融合蛋白和免疫缀合物中，本发明IL-2突变蛋白可以直接或通过接头与另一分子或抗原结合分子连接，且在一些实施方案中，在两者之间包含蛋白水解切割位点。
3.多核苷酸、载体和宿主
本发明提供编码以上任何IL-2突变蛋白或融合物或缀合物的核酸。可以采用本领域熟知的方法，通过从头固相DNA合成或通过PCR诱变编码野生型IL-2的现有序列，产生编码本发明突变蛋白的多核苷酸序列。此外，本发明的多核苷酸和核酸可以包含编码分泌信号肽的区段，并与编码本发明突变蛋白的区段可操作连接，从而可以指导本发明突变蛋白的分泌性表达。
本发明也提供包含本发明核酸的载体。在一个实施方案中，载体是表达载体，例如真核 表达载体。载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。在优选的实施方案中，本发明的表达载体是pYDO_017表达载体。
本发明也提供包含所述核酸或所述载体的宿主细胞。适用于复制并支持突变IL-2蛋白或融合物或免疫缀合物表达的宿主细胞是本领域中公知的。可以用特定的表达载体转染或转导这类细胞，并且可以生长大量的含载体细胞以用于接种大规模发酵罐，从而获得充足量的IL-2突变体或融合物或免疫缀合物用于临床应用。在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞(例如CHO细胞或293细胞)。例如，可以在细菌中生成多肽，尤其在不需要糖基化时。在表达后，可以在可溶性级分中将多肽从细菌细胞糊分离并可以进一步纯化。除了原核生物外，真核微生物如丝状真菌或酵母也是适合编码多肽的载体的克隆或表达宿主，其中包括糖基化途径已被“人源化”的真菌和酵母菌株，这导致生成具有部分或完全的人糖基化模式的多肽。参见Gerngross,NatBiotech22,1409-1414(2004)和Li等,NatBiotech24,210-215(2006)。可用的哺乳动物宿主细胞系的例子是由SV40转化的猴肾CV1系(COS-7)；人胚胎肾系(293或293T细胞，如例如记载于Graham等,JGenVirol36,59(1977))、幼仑鼠肾细胞(BHK)、小鼠塞托利(sertoli)细胞(TM4细胞，如例如记载于Mather,BiolReprod23,243-251(1980))、猴肾细胞(CV1)、非洲绿猴肾细胞(VERO-76)、人宫颈癌细胞(HELA)、犬肾细胞(MDCK)，buffalo大鼠肝细胞(BRL3A)、人肺细胞(W138)、人肝细胞(HepG2)、小鼠乳房肿瘤细胞(MMT060562)、TRI细胞(如例如记载于Mather等,AnnalsN.Y.AcadSci383,44-68(1982))、MRC5细胞和FS4细胞。其它可用的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞，包括dhfr-CHO细胞(Urlaub等,ProcNatlAcadSciUSA77,4216(1980))；和骨髓瘤细胞系如YO、NS0、P3X63和Sp2/0。在一个实施方案中，宿主细胞是真核生物细胞，优选为哺乳动物细胞如中国仓鼠卵巢(CHO)细胞、人胚胎肾(HEK)细胞或淋巴细胞(例如Y0、NS0、Sp20细胞)。
4.制备方法
再一方面，本发明提供制备本发明IL-2突变蛋白或融合物或缀合物的方法，其中所述方法包括，在适合IL-2突变蛋白或融合物或缀合物表达的条件下，培养包含编码所述蛋白或融合物或缀合物的核酸的宿主细胞，如上文所提供的，和任选地从所述宿主细胞(或宿主细胞培养基)回收所述蛋白或融合物或缀合物。
5.测定法
可以通过本领域中已知的多种测定法对本文中提供IL-2突变蛋白进行鉴定，筛选，或表征其物理/化学特性和/或生物学活性。
一方面，可以对本发明的IL-2突变蛋白，测试其与IL-2受体的结合活性。例如，可以通过本领域已知的方法，诸如ELISA，Western印迹等，或本文实施例公开的例示性方法，来测定与人IL-2Rα或β蛋白的结合。例如，可以使用流式细胞术进行测定，其中使经转染在细胞表面上表达突变蛋白的细胞例如酵母展示细胞，与标记的(例如生物素标记的)IL-2Rα或β蛋白进行反应。备选地，突变蛋白与受体的结合，包括结合动力学(例如K D值)，可以使用重组突变蛋白-Fc融合物，在生物膜层干涉(BLI)测定法中测定。在一些实施方案中，使用如实施例所描述的BLI测定法。
再一方面，可以通过测定在受体结合下游发生的信号传导和/或免疫激活效应。来间接测 量IL-2突变蛋白结合IL-2受体的能力。
因此，在一些实施方案中，提供了用于鉴定具有生物学活性的突变IL-2蛋白的测定法。生物学活性可以包括，例如诱导具有IL-2受体的T和/或NK细胞和/或Treg细胞增殖的能力、诱导具有IL-2受体的T和/或NK细胞和/或Treg细胞中IL-2信号传导的能力、降低的诱导T细胞中细胞凋亡的能力、诱导肿瘤消退和/或改善存活的能力、和降低的体内毒性性质，例如降低的血管通透性。本发明也提供体内和/或体外具有这类生物学活性的突变IL-2蛋白。
本领域中公知多种方法可以用于测定IL-2的生物学活性。例如，用于测试本发明IL-2突变蛋白刺激NK细胞生成IFN-γ的能力的合适测定法，可以包括如下步骤：将培养的NK细胞与本发明的突变IL-2蛋白或融合或免疫缀合物温育，并随后通过ELISA测量培养基中的IFN-γ浓度。IL-2信号传导诱导数个信号传导途径，并且牵涉JAK(Janus激酶)和STAT(信号转导物和转录的激活剂)信号传导分子。
IL-2与受体β和γ亚基的相互作用导致受体以及JAK1和JAK3(分别与β和γ亚基结合)的磷酸化。然后，STAT5与磷酸化受体结合，并自身在非常重要的酪氨酸残基上磷酸化。这导致STAT5从受体解离、STAT5二聚化以及STAT5二聚体移位至细胞核，在该处它们促进靶基因的转录。由此，可以例如通过测量STAT5的磷酸化，评估突变体IL-2多肽经由IL-2受体诱导信号传导的能力。此方法的详情已经披露在实施例中。例如，可以将PBMC用本发明的突变体IL-2多肽或融合物或免疫缀合物处理，并通过流式细胞术测定磷酸化STAT5的水平。
此外，可以通过将从血液分离的T细胞或NK细胞与本发明的突变体IL-2多肽或免疫缀合物温育，接着测定经处理细胞的裂解物中的ATP含量，以测量T细胞或NK细胞应答IL-2的增殖。在处理前，可以用植物凝集素(PHA-M)预刺激T细胞。此测定法允许对存活细胞数目的灵敏定量，本领域中还已知大量合适的备选测定法(例如[3H]-胸苷掺入测定法、细胞滴定GloATP测定法、AlamarBlue测定法、WST-1测定法、MTT测定法)。
再有，可以在多种本领域中已知的动物肿瘤模型中评估突变的IL-2对肿瘤生长和存活的影响。例如，可以将人癌症细胞系的异种移植物植入免疫缺陷型小鼠，并用本发明的突变体IL-2多肽或融合物或免疫缀合物处理。可以基于死亡率、生命期观察(不良作用的可见症状，例如行为、体重、体温)以及临床和解剖病理学(例如测量血液化学值和/或组织病理学分析)来测定本发明的突变体IL-2多肽、融合和免疫缀合物在体内的毒性。例如，可以在预处理血管通透性动物模型中用血管渗漏报告分子检查通过用IL-2处理所诱导的血管通透性。优选地，血管渗漏报告分子足够大以揭示用于预处理的IL-2野生型形式的通透性。
此外，对于本发明的IL-2糖基化突变蛋白，糖基化的存在、不存在或程度也可通过本领域的技术人员已知的任何方法来测定，包括分子量(MW)偏移的半定性测量，如通过蛋白质印迹或自考马斯染色的SDS-PAGE凝胶所观察，而定量测量可包括利用质谱仪技术和观察对应于天冬酰胺连接的糖基化的添加的MW偏移，或通过观察由酶诸如肽-N-糖甘酶F(PNGase-F；SigmaAldrich，St.Louis，MO)去除天冬酰胺连接的糖基化而伴有的质量偏移。
6.筛选方法
再一方面，本发明提供用于获得具有改善性质的IL-2突变蛋白的方法。
在一个实施方案中，本发明提供用于获得IL-2突变蛋白的方法，包括如下步骤：
-在IL-2与IL-2Ra的结合界面人为改造一个或多个(如两个或三个)糖基化基序N-X-S/T(X可以为任意氨基酸，但P(脯氨酸)除外)，优选在选自IL-2的如下区域引入糖基化基序：aa35-40,aa41-47，aa62-64,aa68-74，aa74-76；
-使得改造的IL-2突变蛋白，例如以Fc融合物形式(例如FcLALA融合物)，在哺乳动物细胞(例如HEK293或者CHO细胞)中表达。对于引入糖基化基序的位点的设计，N-糖基化预测工具可用于选择可加以突变以促进潜在N-连接糖基化的位点，例如通过鉴定可加以突变以形成标准N-x-T/S糖基化位点(其中N为天冬酰胺，x为除脯氨酸的任何氨基酸)的残基。此外，可以使用基于结构的方法，鉴定IL-2中与IL-2Rα距离为
Figure PCTCN2019107054-appb-000001
并且侧链暴露在溶液中的氨基酸，作为突变为天冬酰胺的候选氨基酸。在一些优选实施方案中，引入的糖基化基序突变选自：K35N-L36-T37；R38N-M39-L40S；T41N-F42-K43S；K43N-F44-Y45T；Y45N-M46-P47S；E62N-L63-K64T；E68N-V69-L70S；L72N-A73-Q74T；Q74N-S75-K76T。
另一方面，本发明提供用于获得IL-2突变蛋白的方法，包括如下步骤：
-在IL-2的B’C’loop环区(aa73-83)引入缺失和/或替代以形成缩短的环区，优选地将其替代为其它四螺旋短链细胞因子家族成员如IL15的B’C’loop环序列以形成B’C’loop嵌合体，或将IL-2的B’C’loop截短以形成B’C’loop截短体，优选地缩短的环区具有小于10，9,8，且优选等于7个氨基酸长度；优选地自环区C端截短1、2、3或4个氨基酸；优选地缩短的环区具有序列A(Q/G)S(K/A)N(F/I)H，或SGDASIH；
-使得改造的IL-2突变蛋白，例如以Fc融合物形式(例如FcLALA融合物)，在哺乳动物细胞(例如HEK293或者CHO细胞)中表达。
在一个实施方案中，所述方法还包括：在蛋白表达和纯化后，鉴定成药性(例如表达量和/或产品稳定性和/或同质性，例如一步Fc亲和层析纯度)改善的IL2突变蛋白。在一个优选实施方案中，在IL-2的区域aa35-40或aa74-76引入糖基化基序突变，以改善突变蛋白的成药性。优选地，引入的糖基化基序突变选自：K35N-L36-T37；R38N-M39-L40S；和Q74N-S75-K76T。在另一优选实施方案中，通过将B’C’loop环替代为缩短的环例如IL15的环序列或通过截短B’C’loop环，以改善突变蛋白的成药性。优选地，缩短后的环序列选自：A(Q/G)S(K/A)NFH，或SGDASIH。在再一优选实施方案中，所述方法包括，在糖基化突变之外，还引入其他点突变，例如K35Q，以改善突变蛋白的成药性。如本领域技术人员明了，可以将这些突变与赋予其它改良性质的突变组合，以获得具有多重改良性质的IL-2突变蛋白。
在一个实施方案中，所述方法还包括：鉴定相对于野生型IL-2表现出降低的(优选消除的)IL-2Ra结合能力的IL-2突变蛋白。在一个实施方案中，IL-2突变蛋白与IL-2Ra的结合能力通过测定亲和力KD值确定，例如通过生物膜薄层干涉技术测定。再一实施方案中，结合能力通过测定IL-2突变蛋白对CD25 +T细胞的激活功效来确定。在一个实施方案中，IL-2突变蛋白，相对于野生型IL-2，表现出降低的CD25 +T细胞激活功效，例如通过测定细胞中p-STAT5信号的激活所确定的。优选地将突变引入IL-2的区域：aa41-47或aa68-70或aa72-74，以形成潜在的N连接糖基化位点，然后检测突变是否导致降低或消除的IL-2与IL-2Rα的结合。优选地，引入的糖基化基序突变选自：T41N-F42-K43S；K43N-F44-Y45T；Y45N-M46-P47S；E68N-V69-L70S；L72N-A73-Q74T。如本领域技术人员明了，可以将这些糖基化突变与赋予其它改良性质的突变组合，以获得具有多重改良性质的IL-2突变蛋白。
在一个实施方案中，所述方法还包括鉴定相对于野生型IL-2表现出具有增强的IL-2Rβ结合的IL-2突变蛋白。在一个实施方案中，IL-2突变蛋白与IL-2Rβ的结合能力通过测定亲和力KD值确定，例如通过生物膜薄层干涉技术测定。再一实施方案中，结合能力通过测定IL-2突变蛋白对CD25 -T细胞的激活功效来确定。在一个实施方案中，IL-2突变蛋白，相对于野生型IL-2，表现出增强的CD25 -T细胞激活功效，例如通过测定细胞中p-STAT5信号的激活所确定的。在一个优选实施方案中，将B’C’loop环替代为缩短的环例如IL15的环序列或通过截短B’C’loop环，以增强对IL-2Rβ的结合。优选地，缩短后的环序列选自：A(Q/G)S(K/A)NFH，或SGDASIH。如本领域技术人员明了，可以将这些糖基化突变与赋予其它改良性质的突变组合，以获得具有多重改良性质的IL-2突变蛋白。
在再一实施方案中，所述方法包括组合引入上述改善成药性的突变、降低IL2Ra结合的突变、和/或增强IL2Rβ结合的突变，和/或赋予其它改良性质的突变组合，以获得具有多重改良性质的IL-2突变蛋白。在一个优选实施方案中，组合引入糖基化突变，例如在区域aa41-47和aa68-74，以及缩短B’C’loop环区长度的截短和/或替代突变。在一个优选实施方案中，所述方法包括鉴定相对于野生型IL-2表现出降低的IL-2Ra结合且增强的IL-2Rβ结合的IL-2突变蛋白，任选地鉴定还具有改善成药性(例如改善的表达和/或纯度、和/或产品稳定性和/或同质性)的IL-2突变蛋白。
在一些实施方案中，用作突变模板的亲本野生型IL-2蛋白优选与SEQ ID NO:26具有至少85％，或至少90％或95％的同一性，更优选地为来源人的IL-2蛋白。
7.药物组合物和药物制剂
本发明还包括包含IL-2突变蛋白或其融合物或免疫缀合物的组合物(包括药物组合物或药物制剂)和包含编码IL-2突变蛋白或其融合物或免疫缀合物的多核苷酸的组合物。这些组合物还可以任选地包含合适的药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。
适用于本发明的药用载体可以是无菌液体，如水和油，包括那些具有石油、动物、植物或合成起源的，如花生油、大豆油、矿物油、芝麻油等。当静脉内施用药物组合物时，水是优选的载体。还可以将盐水溶液和水性右旋糖以及甘油溶液用作液体载体，特别是用于可注射溶液。合适的药用赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、米、面粉、白垩、硅胶、硬脂酸钠、甘油单硬脂酸酯、滑石、氯化钠、干燥的脱脂乳、甘油、丙烯、二醇、水、乙醇等。对于赋形剂的使用及其用途，亦参见“Handbook of PharmaceuticalExcipients”,第五版,R.C.Rowe,P.J.Seskey和S.C.Owen,PharmaceuticalPress,London,Chicago。若期望的话，所述组合物还可以含有少量的润湿剂或乳化剂，或pH缓冲剂。这些组合物可以采用溶液、悬浮液、乳剂、片剂、丸剂、胶囊剂、粉末、持续释放配制剂等的形式。口服配制剂可以包含标准载体，如药用级甘露醇、乳糖、淀粉、硬脂酸镁、糖精。
可以通过将具有所需纯度的本发明的IL-2突变蛋白、融合物或免疫缀合物，与一种或多种任选的药用辅料(Remington′s Pharmaceutical Sciences，第16版，Osol，A.编(1980))混合，来制备包含本发明的药物制剂，优选地以冻干制剂或水溶液的形式。示例性的冻干抗体制剂描述于美国专利号6,267,958。水性抗体制剂包括美国专利号6,171,586和WO2006/044908中所述的那些，后一种制剂包括组氨酸-乙酸盐缓冲剂。此外，可制备持续释放制剂。持续释放 制剂的合适实例包括含有蛋白的固体疏水聚合物的半渗透基质，所述基质呈成形物品，例如薄膜或微囊形式。
本发明的药物组合物或制剂还可以包含一种或多种其它活性成分，所述活性成分是被治疗的特定适应症所需的，优选具有不会不利地影响彼此的互补活性的那些活性成分。例如，理想的是还提供其它抗癌活性成分，例如化疗剂、PD-1轴结合拮抗剂(例如抗PD-1抗体或抗PD-L1抗体或抗PD-L2抗体)。所述活性成分以对于目的用途有效的量合适地组合存在。
因此，在一个实施方案中，组合物还包含第二治疗剂。例如，第二治疗剂可以是免疫检查点抑制剂。例如第二治疗剂可以选自包括但不限于：例如抗-CTLA-4抗体、抗CD47抗体、抗-PD-1抗体、抗-PD-L1抗体、抗-CD40抗体、抗-OX40(亦称为CD134,TNFRSF4,ACT35和/或TXGP1L)抗体、抗-LAG-3抗体、抗-CD73抗体、抗-CD137抗体、抗-CD27抗体、抗-CSF-1R抗体、TLR激动剂或IDO或TGFβ的小分子拮抗剂的一种或多种。优选地，第二治疗剂是PD-1拮抗剂，尤其是抗PD-1抗体，抗PD-L1抗体、抗LAG-3，抗CD47。除了免疫治疗药物，第二治疗剂也可以是其它放疗或化疗药物。
8.组合产品
在一方面，本发明还提供了组合产品，其包含本发明的突变蛋白或其融合物或免疫缀合物，以及一种或多种其它治疗剂(例如化疗剂、其他抗体、细胞毒性剂、疫苗、抗感染活性剂等)。本发明的组合产品可用于本发明的治疗方法中。
在一些实施方案中，本发明提供组合产品，其中所述其它治疗剂为例如有效刺激免疫反应从而进一步增强、刺激或上调受试者的免疫反应的治疗剂如抗体。在一些实施方案中，其它抗体为例如抗PD-1抗体或抗PD-L1抗体或抗PD-L2抗体或抗-LAG-3抗体或抗-CTLA-4抗体或抗TIM-3抗体。
在一些实施方案中，所述组合产品用于预防或治疗肿瘤。在一些实施方案中，肿瘤为癌症，例如胃肠道癌症，例如胃癌、直肠癌、结肠癌、结肠直肠癌等；或皮肤癌，例如黑素瘤；或肾细胞癌、膀胱癌、非小细胞肺癌等。在一些实施方案中，所述组合产品用于预防或治疗感染，例如细菌感染、病毒感染、真菌感染、原生动物感染等。
9.治疗方法和用途
在本文中，术语“个体”或“受试者”可互换地使用，是指哺乳动物。哺乳动物包括但不限于驯化动物(例如，奶牛、绵羊、猫、犬和马)、灵长类(例如，人和非人灵长类如猴)、兔和啮齿类(例如，小鼠和大鼠)。特别地，受试者是人。
在本文中，术语“治疗”指意欲改变正在接受治疗的个体中疾病之天然过程的临床介入。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、减小疾病的任何直接或间接病理学后果、防止转移、降低病情进展速率、改善或缓和疾病状态，以及缓解或改善预后。
在一方面中，本发明提供刺激受试者免疫系统的方法，所述方法包括向所述受试者施用有效量的包含本发明IL-2突变蛋白或融合物或免疫缀合物的药物组合物。本发明IL-2突变蛋白对于CD25 -CD122 +效应细胞(细胞毒性CD8 +T细胞和NK细胞)具有高活性和选择性，并具有降低的对CD25 +Treg细胞的刺激作用。因此，可以以低剂量使用本发明IL-2突变蛋白， 以刺激受试者的免疫系统。
因此，在一些实施方案中，本发明涉及在受试者中增强机体的免疫应答的方法，所述方法包括向所述受试者施用有效量的本文所述的任何IL-2突变蛋白、或其融合物或免疫缀合物。在一些实施方案，将本发明的IL-2突变蛋白或其融合物或免疫缀合物施用于携带肿瘤的受试者，刺激抗肿瘤免疫应答。在另一些实施方案中，将本发明的抗体或其抗原结合部分施用于携带感染的受试者，刺激抗感染免疫应答。在一个实施方案中本发明IL-2突变蛋白可以与Treg耗竭抗体(例如，FcγR介导的Treg耗竭)组合使用，以进一步降低由Treg引起的免疫抑制作用。在一个实施方案中，本发明IL-2突变蛋白可以与免疫检查点抑制剂组合施用，以例如提高癌症免疫治疗效果，例如与抗PD-1和抗CTLA-4组合。
在另一方面中，本发明涉及治疗受试者疾病，如肿瘤和癌症和感染的方法，所述方法包括向所述受试者施用有效量的本文所述的任何IL-2突变蛋白、或其融合物或免疫缀合物。
癌症可以处于早期、中期或晚期或是转移性癌。在一些实施方案中，肿瘤或肿瘤细胞可以选自结直肠肿瘤、卵巢肿瘤、胰腺肿瘤、肺肿瘤、肺肿瘤，肝肿瘤，乳房肿瘤，肾肿瘤，前列腺肿瘤，胃肠肿瘤，黑素瘤，宫颈肿瘤，膀胱肿瘤，成胶质细胞瘤和头颈部肿瘤。在一些实施方案中，癌症可以选自结直肠癌，卵巢癌，胰腺癌，肺癌，肝癌，乳腺癌，肾癌，前列腺癌，胃肠癌，黑素瘤，宫颈癌，膀胱癌，成胶质细胞瘤和头颈癌。在一些实施方案中，肿瘤是黑色素瘤、肾细胞癌、结直肠癌、膀胱癌、非小细胞肺癌。
在另一方面中，本发明涉及治疗受试者感染性疾病，例如慢性感染的方法，所述方法包括向所述受试者施用有效量的本文所述的任何IL-2突变蛋白或其片段，或包含所述抗体或片段的免疫缀合物、多特异性抗体，或药物组合物。在一个实施方案中，所述感染是病毒感染。
在一些实施方案中，除了本发明IL-2突变蛋白或其融合物或缀合物外，本发明的方法还包括向所述受试者联合施用一种或多种疗法(例如治疗方式和/或其它治疗剂)。在一些实施方案中，治疗方式包括手术治疗和/或放射疗法。在一些实施方案中，本发明方法还包括施用至少一种其它的免疫刺激性抗体，例如抗PD-1抗体、抗PD-L1抗体、抗-LAG-3抗体、抗CD43抗体、和/或抗CTLA-4抗体，这些抗体可以是例如全人源的、嵌合的、或人源化的抗体。
在一些实施方案中，抗PD-1抗体选自下组：IBI308(信迪利单抗，WO2017/025016A1)，MDX-1106(nivolumab，OPDIVO)，Merck 3475(MK-3475，pembrolizumab，KEYTRUDA)和CT-011(Pidilizumab)。在一些实施方案中，抗PD-1抗体是MDX-1106。在一些实施方案中，抗PD-1抗体是nivolumab(CAS注册号：946414-94-4)。在进一步的一些实施方案中，单独或与PD-1拮抗剂组合的IL-2突变蛋白或其片段还能与一种或多种其它疗法例如治疗方式和/或其它治疗剂组合施用。在一些实施方案中，治疗方式包括外科手术(例如肿瘤切除术)；放射疗法(例如，外粒子束疗法，它涉及其中设计照射区域的三维适形放射疗法)、局部照射(例如，指向预选靶或器官的照射)或聚焦照射等。
在一些实施方案中，本文提供了治疗疾病(例如，肿瘤)的方法，包括给予受试者本文所述的突变蛋白和CTLA-4拮抗剂抗体。抗-CTLA-4抗体可以是例如选自以下的抗体:
Figure PCTCN2019107054-appb-000002
(ipilimumab或抗体10D1，描述于PCT公开号WO 01/14424),tremelimumab(旧称ticilimumab,CP-675,206)，和描述于以下公开文献中的抗-CTLA-4抗体：WO 98/42752；WO 00/37504；美国专利号6,207,156；Hurwitz等(1998)Proc.Natl.Acad.Sci.USA  95(17):10067-10071；Camacho等(2004)J.Clin.Oncology 22(145):Abstract No.2505(抗体CP-675206)；和Mokyr等(1998)Cancer Res.58:5301-5304。
在一些实施方案中，本文提供了治疗疾病(例如，肿瘤)的方法，包括给予受试者本文所述的抗-突变蛋白和抗-LAG-3拮抗剂抗体。抗-LAG3抗体可以是例如选自以下的抗体：描述于美国专利申请号US2011/0150892和WO2014/008218的抗体25F7,26H10,25E3,8B7,11F2或17E5，或包含这些抗体的CDR或可变区的抗体；BMS-986016；描述于US 2011/007023中的IMP731。
在一些实施方案中，本发明的IL-2突变蛋白可以与化疗或化疗剂联合施用。在一些实施方案中，本发明的IL-2突变蛋白可以与放疗或放疗剂联合施用。在一些实施方案中，本发明的IL-2突变蛋白可以与靶向疗法或靶向治疗剂联合施用。在一些实施方案中，本发明的IL-2突变蛋白可以与免疫疗法或免疫治疗剂，例如单克隆抗体联合施用。
本发明的突变蛋白(以及包含其的药物组合物或其融合物或免疫缀合物，和任选地另外的治疗剂)可以通过任何合适的方法给药，包括肠胃外给药，肺内给药和鼻内给药，并且，如果局部治疗需要，病灶内给药。肠胃外输注包括肌内、静脉内、动脉内、腹膜内或皮下给药。在一定程度上根据用药是短期或长期性而定，可通过任何适合途径，例如通过注射，例如静脉内或皮下注射用药。本文中涵盖各种用药时程，包括，但不限于，单次给药或在多个时间点多次给药、推注给药及脉冲输注。
为了预防或治疗疾病，本发明突变蛋白的合适剂量(当单独或与一种或多种其他的治疗剂组合使用时)将取决于待治疗疾病的类型、抗体的类型、疾病的严重性和进程、以预防目的施用还是以治疗目的施用、以前的治疗、患者的临床病史和对所述抗体的应答，和主治医师的判断力。所述抗体以一次治疗或经过一系列治疗合适地施用于患者。
再一方面，本发明也提供本发明IL-2突变蛋白、组合物、免疫缀合物、融合物在制备用于前述方法(例如用于治疗)的药物中的用途。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限制本发明的保护范围。
实施例1.白细胞介素-2突变体的设计
●白细胞介素-2糖基化蛋白的设计
根据PDB数据库中白细胞介素-2(Interleukin-2，简称IL-2)与其α受体CD25(简称IL-2Rα)的晶体结构(PDB:1Z92)(图1A)，我们通过氨基酸的定点突变，在IL-2与IL-2Ra的结合界面人为改造一个N-X-S/T基序(X可以为任意氨基酸，但P(脯氨酸)除外)，使得IL-2在HEK293或者CHO细胞表达过程中，通过细胞的翻译后修饰，在IL-2的表面形成一个多糖链，阻断IL-2与IL-2R结合(如图1B的结构示意图所示)。
IL-2糖基化位点设计：找出IL-2中与IL-2Ra距离为
Figure PCTCN2019107054-appb-000003
并且侧链暴露在溶液中的氨基酸，将其突变为天冬酰氨，并将后面第三个氨基酸突变丝氨酸或者苏氨酸，形成一个N-X-S/T基序(X可以为任意氨基酸，但P除外)，见表1。
表1.IL-2糖基化蛋白的突变位点
名称 突变氨基酸和N-X-S/T基序
L007(IL-2 glycan1) K35N-L36-T37
L 008(IL-2 glycan2) R38N-M39-L40S
L 009(IL-2 glycan3) E68N-V69-L70S
L 010(IL-2 glycan4) Y45N-M46-P47S
L 011(IL-2 glycan5) K43N-F44-Y45T
L 012(IL-2 glycan6) E62N-L63-K64T
L 013(IL-2 glycan7) L72N-A73-Q74T
L 014(IL-2 glycan8) Q74N-S75-K76T
L 015(IL-2 glycan9) T41N-F42-K43S
●IL-2 B’C’loop嵌合体和截短体的设计
B’C’loop：IL-2的B helix与C helix的连接序列(图2A)，包括A73-R83共11个氨基酸。
比较IL-2单体(PDB:1M47)和复合物的晶体结构(PDB：2ERJ)，我们发现在IL-2单体的晶体结构中B’C’loop是缺失的，这是由于B’C’loop在溶液中非常活跃，无法形成相对稳定的构象。
通过对B’C’loop进行基因工程改造，增加B’C’loop的稳定性，从而增加IL-2的稳定性和与IL-2R的亲和力。因此我们比对了人IL15晶体结构(PDB:2Z3Q)，发现它的B’C’loop较短且稳定(图2.B)。因此我们设计了一个IL-2嵌合分子(L017)和4个截短体分子(L057～L060)(见表2)。
表2.IL-2 B’C’loop优化序列
名称 B’C’loop序列
L 001(IL-2 WT) AQSKNFHLRPR
L 017(IL-2 hyb15BCL) SGDASIH
L 057(IL-2 truncate1) AQSKNFH
L 058(IL-2 truncate2) AGSKNFH
L 059(IL-2 truncate3) AQSANFH
L 060(IL-2 truncate4) AQSANIH
实施例2：IL-2突变体-Fc融合蛋白和IL-2受体的表达纯化
表达质粒的构建
野生型IL-2(uniprot:P60568,aa21-153,C125S，简称IL-2 WT),以及IL-2突变体IL-2 3X(R38D,K43E,E61R)，IL-2 glycans和B’C’loop嵌合体和截短体，通过GSGS连接序列与人IgG1的Fc(L234A,L235A，简称FcLALA，SEQ ID NO:28)连接，并构建到pTT5的载体上，用以表达如下蛋白：
蛋白名称 结构 SEQ ID NOs
Y001 IL-2 WT-GSGS-FcLALA SEQ ID NO:1
Y002 IL-2 .3X-GSGS-FcLALA SEQ ID NO:2
Y007 IL-2 .glycan1-GSGS-FcLALA SEQ ID NO:3
Y008 IL-2 .glycan2-GSGS-FcLALA SEQ ID NO:4
Y009 IL-2 .glycan3-GSGS-FcLALA SEQ ID NO:5
Y010 IL-2 .glycan4-GSGS-FcLALA SEQ ID NO:6
Y011 IL-2 .glycan5-GSGS-FcLALA SEQ ID NO:7
Y012 IL-2 .glycan6-GSGS-FcLALA SEQ ID NO:8
Y013 IL-2 .glycan7-GSGS-FcLALA SEQ ID NO:9
Y014 IL-2 .glycan8-GSGS-FcLALA SEQ ID NO:10
Y015 IL-2 .glycan9-GSGS-FcLALA SEQ ID NO:11
Y017 IL-2 hyb15BCL-GSGS-FcLALA SEQ ID NO:12
Y057 IL-2 truncate1-GSGS-FcLALA SEQ ID NO:20
Y058 IL-2 truncate2-GSGS-FcLALA SEQ ID NO:21
Y059 IL-2 truncate3-GSGS-FcLALA SEQ ID NO:22
Y060 IL-2 truncate4-GSGS-FcLALA SEQ ID NO:23
IL-2 WT,IL-2 3X和L011(IL-2 glycan5)通过2个GGGGS与FcLALA连接，并构建到pCDNA3.1的载体上，用以表达以下蛋白：
蛋白名称 结构 SEQ ID NOs
Y038 IL-2 .glycan5-2*(G4S)-FcLALA SEQ ID NO:13
Y040 IL-2 .3X-2*(G4S)-FcLALA SEQ ID NO:14
Y045 IL-2 WT-2*(G4S)-FcLALA SEQ ID NO:15
在L011(IL-2 glycan5)基础上再增加了1个糖基化位点或增加一个K35Q突变位点(K35Q突变基于前述突变蛋白Y007和蛋白质3D结构而设计)，通过GSGS连接序列与FcLALA连接，并构建到pTT5的载体上；用以表达如下蛋白：
蛋白名称 结构 SEQ ID NOs
Y048 IL-2 glycan5。glycan8-GSGS-FcLALA SEQ ID NO:16
Y049 IL-2 glycan5。glycan1-GSGS-FcLALA SEQ ID NO:17
Y050 IL-2 glycan5。K35Q-GSGS-FcLALA SEQ ID NO:18
将B’C’loop嵌合体(L017)和截短体(L057/058)与糖基化IL-2(L011)组合，通过2个GGGGS与FcLALA连接，并构建到pCDNA3.1的载体上，用以表达以下蛋白：
蛋白名称 结构 SEQ ID NOs
Y056 IL-2 .glycan5.15BCL-2*(G4S)-FcLALA SEQ ID NO:19
Y081 IL-2 .glycan5.truncate1-2*(G4S)-FcLALA SEQ ID NO:24
Y082 IL-2 .glycan5.truncate2-2*(G4S)-FcLALA SEQ ID NO:25
上述蛋白分子的具体序列信息见序列表。
用于构建上述分子的野生型IL-2 WT的序列示于SEQ ID NO:26中，在该序列的125位具有C125S突变，以避免二硫化物桥接的IL-2二聚体形成。IL-2 3X是之前文献(Rodrigo Vazquez-Lombardi等，Nature Communications,8:15373,DOI:10.1038/ncomms15373)中报道的IL-2突变体，与IL-2 WT相同也包含C125S突变，并包含突变R38D,K43E,E61R，其序列示于SEQ ID NO:27中。据文献报道，IL-2 3X与IL-2Rα不结合，与IL-2Rβ的结合力维持与野生型IL-2 相当。
IL-2融合蛋白的表达纯化
根据所需转染体积传代Expi293细胞(Invitrogen)，转染前一天将细胞密度调整至1.5×10 6个细胞/ml。转染当天细胞密度约为3×10 6个细胞/ml。取终体积1/10(v/v)的Opti-MEM培养基(Gibco货号：31985-070)作为转染缓冲液，加入上述构建的表达质粒，混匀，用0.22μm的滤头过滤备用。加合适的聚乙烯亚胺(PEI)(Polysciences，23966)到上一步的质粒中(质粒与PEI的质量比例为1:3)，混匀后室温孵育10min，获得DNA/PEI混合物。将DNA/PEI混合物轻柔倒入HEK293细胞并混匀，在37℃，8％CO 2的条件下培养24h后，补加VPA(Sigma，货号：P4543-100G)使终浓度至2mM及2％(v/v)Feed(1g/L Phytone Peptone+1g/L Difco Select Phytone)，继续培养6天。
细胞培养液以13000rpm离心20min，收集上清，用预装柱Hitrap Mabselect Sure(GE,11-0034-95)纯化上清液。操作如下：纯化前用5倍柱体积的平衡液(20mM Tris,150mM NaCl,pH7.2)平衡填料柱；将收集的上清通过柱子，再用10倍柱体积的平衡液清洗填料柱，去除非特异性结合蛋白；用5倍柱体积的洗脱缓冲液(100mM sodium citrate，pH 3.5)冲洗填料，收集洗脱液。每1ml洗脱液加入80μL Tris(2M Tris)，使用超滤浓缩管(MILLIPORE，货号：UFC901096)交换到PBS缓冲液(Gibco，货号：70011-044)中，并测定浓度。取100μg纯化后蛋白，调整浓度至1mg/mL，使用凝胶过滤色谱柱SW3000(TOSOH货号：18675)测定蛋白纯度。
糖基化突变体Y007，Y008和Y014通过突变表面一个或两个氨基酸后，使蛋白的表达量和纯度相对于Y001都有了明显的提升。Y048，Y049和Y050通过在Y011基础上再增加了1个糖基化位点或增加一个K35Q突变位点，使得表达量从7.77mg/L提高到了50mg/L以上(Y048和Y049)或40mg/L(Y050)，纯度从31.35％提高到80％以上，使得分子的成药性得到了显著的改善。
B’C’loop嵌合体(Y017)和截短体(Y057/058/059)相比于Y001，表达量和一步亲和层析纯度都有很大的提高。
将B’C’loop优化后的序列和糖基化突变L011组合后，与具有L011的突变蛋白Y011相比,Y056,Y081和Y082在表达量和纯度都得到了提升(表3)。
表3.IL-2突变体在HEK293的表达量和纯度
Figure PCTCN2019107054-appb-000004
Figure PCTCN2019107054-appb-000005
●IL-2受体的表达和纯化
人IL-2受体 Uiprot:P01589，aa-217)和(Uiprot:P14784，aa27-240)在序列的C末端接上avi标签(一段多肽：GLNDIFEAQKIEWHE，可以被BirA酶催化发生生物素化)和6个组氨酸标签(HHHHHH)，分别构建到pTT5载体上。质粒转染293F细胞(Invitrogen)方法同IL-2Fc融合蛋白的表达方法。
纯化前将收集的培养基4500rpm离心30min，弃掉细胞。再将上清使用0.22μl的滤器过滤。将纯化使用的镍柱(5ml Histrap excel,GE,17-3712-06)用0.1M NaOH浸泡2h，然后用5-10倍柱体积的超纯水冲洗，去除碱液。纯化前用5倍柱体积的结合缓冲液(20mM Tris pH 7.4,300mM NaCl)平衡纯化柱；将细胞上清通过平衡后的柱子；用10倍柱体积的冲洗缓冲液(20mM Tris 7.4,300mM NaCl,10mM imidazole)通过柱子，去除非特异性结合的杂蛋白；然后用3-5倍柱体积洗脱液(20mM Tris 7.4,300mM NaCl,100mM imidazole)将目标蛋白洗脱下来。将收集的蛋白超滤浓缩交换到PBS(Gibco，70011-044)中，然后用superdex200 increase(GE,10/300GL,10245605)进一步分离纯化，收集单体的洗脱峰，柱子的平衡和洗脱缓冲液为PBS(Gibco，70011-044)。取100μg纯化后的蛋白样品，使用凝胶过滤色谱柱SW3000(TOSOH货号：18675)测定蛋白纯度(图3和4)。
实施例3.IL-2突变体Fc融合蛋白(简称：IL-2 mutant-FC)与其受体的亲和力测定
采用生物膜薄层干涉(BiolayerInterferometry，BLI)技术测定本发明IL-2 mutant-FC结合人IL-2Rα和IL-2Rβ的平衡解离常数(KD)。BLI法亲和力测定按照现有的方法(Estep,P等人，High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs,2013.5(2):第270-8页)进行。
实验开始前半个小时，根据样品数量，取合适数量的AHC(ForteBio，18-5060)(用于阳性对照检测)传感器浸泡于SD buffer(PBS 1×，BSA 0.1％，Tween-20 0.05％)中。
取100μl的SD缓冲液、IL-2 mutant-FC、IL-2受体或分别加入到96孔黑色聚苯乙烯半量微孔板(Greiner，675076)中。根据样品位置布板，选择传感器位置。仪器设置参数如下： 运行步骤：Baseline、Loading～1nm、Baseline、Association和Dissociation；各个步骤运行时间取决于样品结合和解离速度，转速为400rpm，温度为30℃。使用ForteBio分析软件分析K D值。
表4a.IL-2 glycan-FC与IL-2R的亲和力K D值
蛋白名称 与IL-2R的亲和力(affinity)(M)
Y001 1.12E-08
Y002 N.B.
Y007 2.55E-09
Y008 4.23E-08
Y009 N.B.
Y010 N.B.
Y011 N.B.
Y012 9.22E-08
Y013 N.B.
Y014 1.03E-08
Y015 N.B.
表4b.IL-2B’C’loop突变体与IL-2R的亲和力K D值
蛋白名称 与IL-2R的亲和力(affinity)(M)
Y001 N.B.
Y017 8.87E-08
Y057 2.34E-07
Y058 3.44E-07
Y059 1.46E-07
Y060 7.63E-07
表4c.IL-2突变组合体与受体的亲和力K D值
蛋白名称 与IL-2R的亲和力(affinity)(M) 与IL-2R的亲和力(affinity)(M)
Y038 N.B N.B
Y040 N.B N.B
Y045 5.03E-08 N.B
Y056 N.B 1.20E-07
Y081 N.B 2.60E-07
Y082 N.B P.F
N.B.:IL-2与受体没有结合；P.F:结合很弱，拟合效果差。；
由以上亲和力数据可知：1)Y009，Y010，Y011，Y013和Y015可以阻断IL-2R的结合(表4a)；2)B’C’loop嵌合分子和截短分子，不仅增加了分子的表达量，还增加分子与IL-2R的亲和力(表4b)；3)IL-2糖基化与B’C’loop改造的组合体Y056和Y081，与Y045(IL-2 WT-2*(G4S)-FcLALA)、Y040(IL-2 .3X-2*(G4S)-FcLALA)和Y038(IL-2 .glycan5-2*(G4S)-FcLALA)相比，在阻断IL2R结合的同时，增强了与IL2R的亲和力。
实施例4：IL-2 mutant-FC体外功能实验
IL-2 WT与IL-2Rα亲和力高于IL-2Rβ和IL-2Rγ，会优先结合细胞表面的IL-2Rα，再招募IL-2Rβγ，通过IL-2Rβγ释放下游p-STAT5信号，刺激T细胞与NK细胞增殖。由于Treg细胞表面有IL-2Rα，效应T细胞与NK细胞表面没有IL-2Rα，正常情况下IL-2 WT会优先刺激Treg细胞增殖，下调免疫反应。IL-2 mutant与IL-2Rα不结合，消除了优先刺激Treg细胞增殖的偏好，同时刺激T细胞与NK细胞增殖，使得效应T细胞与NK细胞的数量有效增加，提高抗肿瘤效应。
本实施例通过检测各IL-2 mutant-FC对原代人CD8 +T细胞p-STAT5信号的激活，验证各突变体对CD25 +细胞激活偏向性的去除，并筛选对CD25 -细胞激活作用较强的突变体。具体步骤如下：
1.复苏PBMC细胞：
a)从液氮取出PBMC细胞(Allcells货号：PB005F，100M装)，迅速置于37℃水浴锅中，复苏PBMC细胞；
b)将细胞加入10mL已预热的、含5％人AB血清(GemCell货号：100-512)与1‰DNA酶(STRMCELL货号：07900)的X-VIVO15(Lonza货号：04-418Q)培养基中，400G、25℃离心10分钟(后续离心均为此条件)洗涤一次；
c)加入20mL培养基重悬细胞，37℃二氧化碳培养箱静置培养过夜。
2.纯化人CD8 +T细胞：
a)吸取步骤1中悬浮细胞液，离心弃上清；
b)加入1mL Robosep缓冲液(STEMCELL货号：20104)与100μL人AB血清及100μL人CD8 +T细胞纯化试剂盒(Invitrogen货号：11348D)中负性筛选抗体混合液重悬细胞；
c)混匀后，4℃孵育20分钟，每5分钟摇晃一次；
d)孵育后，加入10mL Robosep缓冲液，离心洗涤两次；
e)同时，取1mL磁性微球(人CD8 +T细胞纯化试剂盒)，加入7mL Robosep缓冲液置于磁力架上1分钟弃上清，预洗磁性微球；
f)各加入1mL Robosep缓冲液分别重悬微球与细胞，混匀后室温旋转孵育30分钟；
g)孵育后，加入6mL Robosep缓冲液，置于磁力架上1分钟，收集上清；
h)收集液再次置于磁力架上1分钟，收集上清；
i)离心弃上清，使用预热的T培养基重悬，调整密度至1×10 6/mL；
j)取1/3细胞待后需刺激CD25表达，其余细胞置于37℃二氧化碳培养箱静置过夜培养。
3.刺激CD8 +T细胞表达CD25：
a)取1/3步骤2中纯化后的CD8 +T细胞，加入抗人CD3/CD28抗体的磁性微球(GIBCO货号：11131D)，细胞与微球的比例为3:1；
b)置于37℃二氧化碳培养箱静置三天；
c)加入10mL培养基清洗2次；
d)加入培养基调整细胞密度至1×10 6/mL，37℃二氧化碳培养箱中静置培养2天。
4.检测细胞纯度与表达水平：
a)使用抗人CD8-PE(Invitrogen货号：12-0086-42)、抗人CD25-PE(eBioscience货号：12-0259-42)、同型对照抗体(BD货号：556653)检测细胞的CD8与CD25；
b)步骤2中细胞为CD8 +CD25 -T细胞，步骤3中细胞为CD8 +CD25 +T细胞。
5.检测各IL-2 mutant-FC对CD8 +CD25 -T细胞激活p-STAT5信号的EC 50：
a)取CD8 +CD25 -T细胞，以每孔1×10 5细胞铺96孔U底培养板(Costar货号：CLS3799-50EA)；
b)加入100μL各IL-2 mutant-FC、商品化IL-2(R&D货号：202-IL-500)、IL-2 WT-FC、IL-2 3X-FC，最高浓度从266.7nM开始4倍梯度稀释共12个梯度，37℃培养箱中孵育20分钟；
c)加入取55.5μL 4.2％甲醛溶液，室温固定10分钟；
d)离心弃上清，加入200μL冰甲醇(Fisher货号：A452-4)重悬细胞，4℃冰箱孵育30分钟；
e)离心弃上清，用200μL染色缓冲液(BD货号：554657)洗涤3次；
f)加入200μL含抗p-STAT5-AlexFlour647(BD货号：562076，1:200稀释)的破膜/固定缓冲液(BD货号：51-2091KZ)，室温避光孵育3小时；
g)使用染色缓冲液洗涤三次，100μL染色缓冲液重悬细胞，进行流式细胞仪检测；
h)以IL-2分子的浓度为横坐标、AlexFlour647中间荧光度值为纵坐标，制作p-STAT5信号的EC 50值，结果如图5和表5所示。
6.检测各IL-2 mutant-FC对CD8 +CD25 +T细胞激活p-STAT5信号的EC 50：
a)取CD8 +CD25 +T细胞，以每孔1×10 5细胞铺96孔U底培养板；
b)同步骤5中b-h，制作p-STAT5信号的EC 50值，结果如图5和表5所示。
表5.IL-2突变体对CD25 +/-T细胞激活p-STAT5信号的EC 50及其比值(donor2)
Donor2 R&D IL2 Y045 Y040 Y056 Y081
CD25 +pSTAT5 EC 50 0.005086 0.0282 4.12 0.06085 2.186
CD25_pSTAT5 EC 50 0.5945 13.17 34.61 2.856 7.203
CD25 -EC 50/CD25 +EC 50倍数 116.8895 467.0213 8.4005 46.9351 3.2951
实验结果表明(在同一个donor下对比)：
1)对比Y001(IL-2 WT-GSGS-FcLALA)和Y045(IL-2 WT-2*(G4S)-FcLALA)，Y002(IL-2 .3X-GSGS-FcLALA)和Y040(IL-2 .3X-2*(G4S)-FcLALA)，Y011(IL-2 .glycan5-GSGS-FcLALA)和Y038(IL-2 .glycan5-2*(G4S)-FcLALA)的曲线位置，发现长的连接序列(GGGGSGGGGS)优于短的连接序列(GSGS)对于CD25 -CD8 +T细胞的激活(图5A)。
2)嵌合人IL-15的B’C’loop后，Y017(IL-2 hyb15BCL-GSGS-FcLALA)对CD25 -CD8 +T细胞的激活(Y017的EC50值为0.9902)比Y001(EC 50值为10.69)提高了10.79倍(图5A)；而对CD25 +CD8 +T细胞的激活(Y017的EC50值为0.0018)与Y001(EC 50值为0.0020)相当(图5B)。
3)在IL-2的界面增加了一个N聚糖后(Y038)，对CD25 -CD8 +T细胞的激活(Y038的EC 50值为369.0)比野生型的IL-2(Y045，EC 50值为31.73)下降了11.63倍，但优于文献报道的IL-2 3X(Y040)；而在此基础上嵌合人IL-15的B’C’loop后(Y056,EC 50值为8.571)，对CD25 -CD8 +T细胞的激活比Y045提高了3.7倍，比Y038提高了43.05倍(图5C)。
4)在同一个donor下，在对比刺激前后CD8+T细胞，说明Y056和Y081增强了CD25 -CD8 +T细胞的激活，同时减少了对CD25 +细胞激活偏向性(图5D.E和表5)。
序列表
Figure PCTCN2019107054-appb-000006
Figure PCTCN2019107054-appb-000007
Figure PCTCN2019107054-appb-000008
Figure PCTCN2019107054-appb-000009
Figure PCTCN2019107054-appb-000010

Claims (29)

  1. 一种IL-2突变蛋白，其中所述突变蛋白，与野生型IL-2(优选人IL-2，更优选包含SEQ ID NO:26序列的IL-2)相比，包含至少一个突变，所述突变在选自以下的氨基酸位置引入一个或多个糖基化基序N-X-S/T：
    35N-36X-37T/S；38N-39X-40T/S；41N-42X-43T/S；43N-44X-45T/S；45N-46X-47T/S；62N-63X-64T/S；68N-69X-70T/S；72N-73X-74T/S；74N-75X-76T/S，其中，X是除脯氨酸外的任何氨基酸，优选地X是与野生型IL-2相应位置的氨基酸相同的氨基酸，或是其保守取代残基；
    其中氨基酸位置根据SEQ ID NO:26编号。
  2. 权利要求1的突变蛋白，其中，所述突变蛋白包含一个或多个选自以下的突变糖基化基序：35N-36X-37T/S；38N-39X-40T/S；和74N-75X-76T/S，其中氨基酸位置根据SEQ ID NO:26编号，
    其中，与野生型IL-2相比，所述突变蛋白，当在哺乳动物细胞中表达时，优选地以Fc融合蛋白形式表达时，具有改善的表达和/或纯度(优选地，通过在表达和一步亲和纯化后测定突变蛋白的纯度)。
  3. 权利要求2的突变蛋白，其中，与野生型IL-2相比，所述突变蛋白包含选自以下的突变糖基化基序：
    (i)K35N-L36-T37；
    (ii)R38N-M39-L40S；
    (iii)Q74N-S75-K76T；优选所述突变蛋白包含突变糖基化基序K35N-L36-T37。
  4. 根据权利要求2和3的突变蛋白，其中，所述突变蛋白包含与选自以下的氨基酸序列具有至少90％、92％、93％、94％、95％、96％、97％、或98％的同一性的序列：SEQ ID NO:31，32和38。
  5. 根据权利要求1的突变蛋白，其中所述突变蛋白包含一个或多个选自以下的突变糖基化基序：41N-42X-43T/S；43N-44X-45T/S；45N-46X-47T/S；68N-69X-70T/S；72N-73X-74T/S，优选糖基化基序43N-44X-45T/S，其中氨基酸位置根据SEQ ID NO:26编号，
    其中，与野生型IL-2相比，所述突变蛋白具有降低的或消除的IL-2Rα结合。
  6. 权利要求5的突变蛋白，其中，与野生型IL-2相比，所述突变蛋白包含选自以下的突变糖基化基序：
    (i)T41N-F42-K43S；
    (ii)K43N-F44-Y45T；
    (iii)Y45N-M46-P47S；
    (iv)E68N-V69-L70S；
    (v)L72N-A73-Q74T；
    优选地，所述突变蛋白包含突变糖基化基序K43N-F44-Y45T。
  7. 权利要求5-6的突变蛋白，其中，所述突变蛋白还包含：
    (i)选自35N-36X-37T/S；38N-39X-40T/S；和74N-75X-76T/S的突变糖基化基序；和/或
    (ii)突变K35Q，
    其中，与野生型IL-2相比，所述突变蛋白具有降低的或消除的IL-2Rα结合，且在以Fc融合蛋白形式在哺乳动物细胞中表达时具有改善的表达和纯度。
  8. 根据权利要求5-7的突变蛋白，其中，所述突变蛋白包含与选自以下的氨基酸序列具有至少90％、92％、93％、94％、95％、96％、97％、或98％同一性的序列：SEQ ID NO:33,34,35,37，39，和45-47。
  9. 一种IL-2突变蛋白，其中所述突变蛋白，与野生型IL-2(优选人IL-2，更优选包含SEQ ID NO:26序列的IL-2)相比，包含缩短的B’C’环区(即，氨基酸残基aa72和aa84之间的连接序列)，优选地，所述缩短的环区具有小于10，9,8，7，6，或5个的氨基酸长度，且优选7个氨基酸长度，其中氨基酸残基根据SEQ ID NO:26编号。
  10. 权利要求9的突变蛋白，其中所述突变蛋白，相对于野生型IL-2，包含
    (i)对aa73至aa83序列的替代，例如替代为来自四螺旋短链细胞因子IL家族成员的短B’C’环序列，如IL15的B’C’环序列，优选地经替代的环区具有序列SGDASIH；或
    (ii)对aa73至aa83序列的截短，例如自C端截短1、2、3或4个氨基酸；优选地截短的环区具有序列A(Q/G)S(K/A)N(F/I)H，优选所述截短的环区具有序列AQSKNFH或AGSKNFH。
  11. 权利要求8-10的突变蛋白，其中所述突变蛋白，相对于野生型IL-2，具有增强的IL-2Rβ结合，和/或改善的表达产量和/或纯度。
  12. 权利要求8-11的突变蛋白，其中所述突变蛋白包含与选自以下的氨基酸序列具有至少90％、92％、93％、94％、95％、96％、97％、或98％同一性的序列：SEQ ID NO:40-44，优选SEQ ID NO:40-42，更优选SEQ ID NO:40或41。
  13. 一种IL-2突变蛋白，其中所述突变蛋白，与野生型IL-2(优选人IL-2，更优选包含SEQ ID NO:26序列的IL-2)相比，包含组合突变：(i)选自41N-42X-43T/S；43N-44X-45T/S；45N-46X-47T/S；68N-69X-70T/S；72N-73X-74T/S的突变糖基化基序；和(ii)在氨基酸位置aa72至aa84之间的、选自SGDASIH和A(Q/G)S(K/A)N(F/I)H的缩短B’C’环区序列，其中氨基酸位置根据SEQ ID NO:26编号。
  14. 权利要求13的突变蛋白，其中所述突变蛋白包含与选自以下的氨基酸序列具有至少90％、92％、93％、94％、95％、96％、97％、或98％的同一性的序列：SEQ ID NO:48，49或50，优选SEQ ID NO:48或49。
  15. 权利要求1-14的突变蛋白，其中所述突变蛋白，与野生型IL-2相比，具有以下特性之一或多项：
    -具有消除或降低的对IL-2Rα受体的结合亲合力，
    -具有增强的对IL-2Rβ受体的结合亲合力；
    -具有降低的对高亲合力IL-2R受体(IL-2Rαβγ)的结合亲合力；
    -具有增加的对中等亲合力IL-2R受体(IL-2Rβγ)的结合亲合力；
    -降低对CD25+细胞(尤其是CD8+T细胞，特别是Treg细胞)的激活；
    -降低对CD25+细胞(尤其是CD8+T细胞)中IL-2介导的信号传导的刺激作用；
    -去除或降低IL-2优先激活CD25+细胞(尤其是Treg细胞)的偏向性；
    -降低由IL-2诱导的Treg细胞引起的免疫反应下调作用；
    -保持或增强对CD25-细胞的激活作用，
    -刺激效应细胞T细胞和NK细胞的增殖和激活；
    -提高抗肿瘤效应。
  16. 根据权利要求1-15所述的IL-2突变蛋白，其中所述突变蛋白在哺乳动物细胞如HEK293细胞中表达时，具有以下一个或多个特征:
    -优于野生型IL-2蛋白的表达量；
    -优于野生型IL-2蛋白的稳定性；
    -易于纯化至更高的蛋白纯度，例如，一步亲和层析纯化后更高的纯度。
  17. 根据权利要求1-16中任一项所述的IL-2突变蛋白，其中所述突变蛋白与野生型IL-2相比具有至少85％,至少95％,或至少96％的同一性。
  18. 根据权利要求1-16中任一项所述的IL-2突变蛋白，其中所述突变蛋白，与野生型IL-2相比，具有降低的优先刺激CD25+T细胞中p-STATA5信号传导的偏向性，且具有增强的刺激CD25-T细胞中信号传导的能力，
    优选地所述突变蛋白包含组合突变：
    (i)在氨基酸位置43-45的突变糖基化基序K43N-F44-Y45T和在氨基酸位置aa72至aa84之间的替代序列SGDASIH；或
    (ii)在氨基酸位置43-45的突变糖基化基序K43N-F44-Y45T和在氨基酸位置aa72至aa84之间的截短序列AQSKNFH，
    更优选，所述突变蛋白包含SEQ ID NO:48或49的序列，或与其具有至少95％,96％、或更高的同一性的序列。
  19. 一种IL-2突变蛋白融合蛋白，其包含根据权利要求1-18的IL2突变蛋白，优选地与Fc抗体片段融合，优选地IL-2突变蛋白通过接头与Fc融合，所述接头优选为GSGS，更优选为2x(G4S)，
    优选地，所述融合蛋白包含与选自以下的氨基酸序列至少85％，至少95％,或至少96％的同一性：SEQ ID NO:3-13和16-25。
  20. 一种免疫缀合物，其包含权利要求1-18的IL2突变蛋白和抗原结合分子，优选地，抗原结合分子是免疫球蛋白分子，特别是IgG分子，或抗体或抗体片段，特别是Fab分子和scFv分子。
  21. 权要求20的免疫缀合物，其中所述抗原结合分子特异性结合肿瘤细胞上或肿瘤环境中呈现的抗原，例如选自以下的抗原：成纤维细胞活化蛋白(FAP)、生腱蛋白C的A1域(TNC A1)、生腱蛋白C的A2域(TNC A2)、纤连蛋白的外域B(Extra Domain B,EDB)、癌胚抗原(CEA)、和黑素瘤有关的硫酸软骨素蛋白聚糖(MCSP)。
  22. 一种分离的多核苷酸，其编码权利要求1-18的IL-2突变蛋白或权利要求19的融合物或权利要求20-21的免疫缀合物。
  23. 一种表达载体，其包含权利要求22的多核苷酸。
  24. 一种宿主细胞，其包含权利要求22的多核苷酸或权利要求23的载体，优选所述宿主细胞是哺乳动物细胞，特别是HEK293细胞，和酵母。
  25. 一种生产IL-2突变蛋白或其融合物或免疫缀合物的方法，包括在适于表达所述IL-2突变蛋白或融合物或缀合物的条件下培养权利要求24的宿主细胞。
  26. 一种药物组合物，其包含权利要求1-18的IL-2突变蛋白或权利要求19的融合物或权利要求20-21的免疫缀合物和药学可接受载体。
  27. 一种治疗受试者疾病的方法，所述方法包括向所述受试者施用权利要求1-18的IL-2突变蛋白或权利要求19的融合物或权利要求20-21的免疫缀合物或权利要求26的药物组合物，优选地，所述疾病是癌症。
  28. 一种刺激受试者免疫系统的方法，所述方法包括向所述受试者施用有效量的包含权利要求1-18的IL-2突变蛋白或权利要求19的融合物或权利要求20-21的免疫缀合物的药物组合物。
  29. 一种用于获得IL-2突变蛋白的方法，包括如下步骤：
    -在IL-2与IL-2Ra的结合界面通过突变引入一个或多个(如两个或三个)糖基化基序N-X-S/T，其中X可以为任意氨基酸，但P(脯氨酸)除外，和/或在IL-2的B’C’环区通过突变以缩短该环区序列，
    优选地引入如权利要求1-7中描述的糖基化突变和/或如权利要求9-10中描述的B’C’环序列突变，更优选地引入权利要求13或18中描述的组合突变；
    -在哺乳动物细胞(例如HEK293或者CHO细胞)中表达IL-2突变蛋白，例如以Fc融合物形式(例如FcLALA融合物)；
    -鉴定具有如下一项或多项改善性质的突变蛋白：(i)表达量和/或稳定性；(ii)降低IL2Rα结合；(iii)增强的IL2Rβ结合。
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